Characterisation of gene function using double stranded RNA inhibition

ABSTRACT

There is provided a method of identifying DNA responsible for conferring a particular phenotype in a cell which method comprises a) constructing a cDNA or genomic library of the DNA of said cell in a suitable vector in an orientation relative to a promoter(s) capable of initiating transcription of said cDNA or DNA to double stranded (ds) RNA upon binding of an appropriate transcription factor to said promoter(s), b) introducing said library into one or more of said cells comprising said transcription factor, and c) identifying and isolating a particular phenotype of said cell comprising said library and identifying the DNA or cDNA fragment from said library responsible for conferring said phenotype. Using this technique it is also possible to assign function to a known DNA sequence by a) identifying a homologue(s) of said DNA sequence in a cell, b) isolating the relevant DNA homologue(s) or a fragment thereof from said cell, c) cloning said homologue or fragment thereof into an appropriate vector in an orientation relative to a suitable promoter(s) capable of initiating transcription of dsRNA from said DNA homologue or fragment upon binding of an appropriate transcription factor to said promoter(s) and d) introducing said vector into said cell from step a) comprising said transcription factor.

[0001] The present invention is concerned with characterization oridentification of gene function using double stranded RNA inhibition(dsRNAi) and methods of identifying DNA responsible for inducing aspecific phenotype in a cell and a method of assigning function to knowngene sequences.

[0002] It has recently been described in Nature Vol 391, pp.806-811,February 98, that introducing double stranded RNA into a cell results inpotent and specific interference with expression of endogenous genes inthe cell and which interference is substantially more effective thanproviding either RNA strand individually as proposed in antisensetechnology. This specific reduction of the activity of the gene was alsofound to occur in the nematode worm Caenorhabditis elegans (C. elegans)when the RNA was introduced into the genome or body cavity of the worm.

[0003] The present inventors have utilized this technique and applied itfurther to devise novel and inventive methods of assigning functions togenes or DNA fragments, which have been sequenced in various projects,such as, for example, the human genome project and which have yet to beaccorded a particular function and for use in identifying DNAresponsible for conferring a particular phenotype.

[0004] Therefore, according to a first aspect of the present inventionthere is provided a method of identifying DNA responsible for conferringa phenotype in a cell which method comprises a) constructing a cDNA orgenomic library of the DNA of said cell in an orientation relative to apromoter(s) capable of promoting transcription of said cDNA or DNA todouble stranded (ds) RNA upon binding of an appropriate transcriptionfactor to said promoter(s), b) introducing said library into one or moreof said cells comprising said transcription factor, and c) identifyingand isolating a desired phenotype of said cell comprising said libraryand identifying the DNA or cDNA fragment from said library responsiblefor conferring said phenotype.

[0005] In a preferred embodiment of the invention the library may beorganised into hierarchical pools as described in more detail in theexamples provided, prior to step b) such as to include, for example,gene families.

[0006] According to a further aspect of the invention there is alsoprovided a method of assigning function to a known DNA sequence whichmethod comprises a) identifying a homologue(s) of said DNA in a cell, b)isolating the relevant DNA homologue(s) or a fragment thereof from saidcell, c) cloning said homologue or fragment into an appropriate vectorin an orientation relative to a promoter(s) capable of promotingtranscription of dsRNA upon binding of an appropriate transcriptionfactor to said promoters, d) introducing said vector into said cell fromstep a) comprising said transcription factor, and e) identifying thephenotype of said cell compared to wild type.

[0007] In each aspect of the invention, the nucleotide or DNA sequencemay either be provided in a sense and an antisense orientation relativeto a single promoter which has the properties defined above, oralternatively it may be provided between two identical promoters. Inboth embodiments dsRNA is provided from the transcription initiated fromthe promoter following binding of its appropriate transcription factor.

[0008] The cell according to the invention may be derived from orcontained in an organism. Where the cell is contained within anorganism, the organism may be adapted to express the appropriatetranscription factor. The organism may be any of a plant, animal, fungusor yeast but preferably may be the nematode worm C. elegans, which maybe any of a wild type, a nuc-1 or pha-ts mutant of C. elegans or acombination of said mutations. In an alternative embodiment the DNA orcDNA library or the DNA homologue or fragment thereof may,advantageously, be transfected or transformed into a microorganism, suchas a bacterial or yeast cell, which may be fed to the organism, which ispreferably the nematode worm C. elegans. In this embodiment of theinvention the microorganism may be adapted to express the appropriatetranscription factor. Preferably, the microorganism is E. coli.

[0009] In each aspect of the invention, the DNA library, DNA homologueor DNA fragment may be constructed in a suitable DNA vector whichcomprises a sequence of nucleotides which encode said transcriptionfactor. Alternatively, said transcription factor is encoded by a furthervector. In an even further alternative, the cell or organism may expressor be adapted to express said transcription factor. Preferably, any ofthe vectors used in the method according to the invention comprises aselectable marker which may be, for example, a nucleotide sequenceencoding sup-35 or a fragment thereof. The nucleotide sequence may beorientated relative to a promoter such that binding of a transcriptionfactor to the promoter initiates transcription of the DNA into doublestranded RNA. FIG. 10 illustrates the vectors and the orientation of theDNA sequence which enable double stranded RNA production in C. elegans.Thus in one embodiment the DNA is located between two promoters on avector capable of expressing dsRNA upon binding of an appropriatetranscription factor to said promoters. Alternatively, the vectorcomprises two copies of the DNA sequence organised in a sense andantisense orientation relative to the promoter and which marker isselectable when contained in a pha-1 mutant C. elegans. Preferably, thepromoters are any of T7, T3 or SP6 promoters and the transcriptionfactor comprises the appropriate polymerase.

[0010] Preferably, the selectable marker comprises a nucleotide sequencecapable of inhibiting or preventing expression of a gene in said celland which gene is responsible for conferring a known phenotype. Thisnucleotide sequence may be part of or identical to said gene conferringsaid phenotype, and which nucleotide sequence is itself orientedrelative to a suitable promoter(s) capable of initiating transcriptionof double stranded RNA upon binding of an appropriate transcriptionfactor to said promoter(s). Alternatively, the nucleotide sequence maybe a part of or identical to said gene sequence conferring saidphenotype, and which nucleotide sequence is such as to permitintegration of said suitable or further vector by homologousrecombination in the genome of said cell and following said integrationsaid nucleotide sequence is capable of inhibiting expression of saidgene sequence conferring said phenotype. In this embodiment saidnucleotide sequence comprises stop codons sufficient to preventtranslation of said nucleotide sequence following its integration intosaid genome.

[0011] Compounds can, advantageously, in said method be added to saidcell or organism for the purposes of screening for desired phenotypes,such as for example, resistance or sensitivity to the compound whencompared to wild type. The promoters are preferably inducible. Thetranscription factor may in some embodiments be phage derived, such asfor example, a T7 polymerase driven by a phage promoter. However, whenC. elegans is utilised a worm specific or tissue specific promoter canbe used, such as for example, let858, SERCA, UL6, myo-2 or myo-3.Preferably, the E. coil strain is an RNAaseIII and even more preferablyan Rnase negative strain.

[0012] A further aspect of the present invention provides a method ofgenerating a transgenic non-human organism comprising an exogenoustranscription factor and a transgene comprising a promoter operablylinked to DNA fragment which is expressed upon binding of saidtranscription factor thereto, the method comprising a) providing a firsttransgenic organism comprising a first construct incorporating DNAencoding an exogenous transcription factor and a second transgenicorganism comprising a second construct including at least one promoteroperably linked to a desired DNA sequence which is expressed uponbinding of the transcription factor of said first transgenic organismthereto b) crossing said first and second transgenic organisms andselecting offspring expressing said desired DNA sequence. In oneembodiment said first and second transgenic organisms are generated bytransforming said first and second constructs into respectivemicroorganisms for subsequent feeding to the respective organism.Preferably, said second construct comprises said desired DNA sequence inan orientation relative to said promoter so as to be capable ofinitiating transcription of said DNA to dsRNA upon binding of saidtranscription factor thereto. In this embodiment said second constructcomprises two promoters flanking said desired DNA sequence whichpromoters can initiate transcription of said DNA sequence to dsRNA uponbinding of said transcription factor to said promoters. Alternatively,said DNA sequence is provided in a sense and an antisense orientationrelative to said promoter so as to produce dsRNA upon binding of thetranscription factor to the promoters. In each of these embodiments thefirst and/or second constructs may preferably be provided with areporter gene operably linked to a promoter which is capable ofinitiating transcription of said reporter upon binding of saidtranscription factor thereto. Preferably, the reporter gene encodes anyof Luciferase, Green Fluorescent protein, β galactosidase orβ-lactamase.

[0013] The present invention also includes a method of validating clonesidentified in yeast two hybrid vector experiments which experiments arewell known to those skilled in the art and which experiments were firstproposed by Chien et al. (1991) to detect protein—protein interactions.The method according to the invention comprises providing a constructincluding the DNA encoding a protein identified in a two hybrid vectorexperiment, which construct is such that said DNA is provided in anorientation relative to one or more promoters capable of promotingtranscription of said DNA to double stranded RNA upon binding of anappropriate transcription factor to said promoters, transforming a cell,such as a bacterial cell or alternatively transforming an organismcomprising said transcription factor with said constructs andidentifying a phenotypic change in said cell or organism, which may beC. elegans or the like, compared to wild type. Preferably, thetranscription factor is inducible in the cell or organism. Once againthe DNA sequence may be located between two promoters or in both a senseand antisense orientation relative to a single promoter, as describedabove. Preferably, the promoter is a phage polymerase promoter and saidtranscription factor is a RNA polymerase, and preferably T7 polymerases.Also encompassed with the scope of the present invention are vectorsused to transform said cells or organisms and the cells or organismsthemselves.

[0014] In a further aspect of the present invention there is provided amethod of alleviating pest infestation of plants, which method comprisesa) identifying a DNA sequence from said pest which is critical eitherfor its survival, growth, proliferation or reproduction, b) cloning saidsequence from step a) or a fragment thereof in a suitable vectorrelative to one or more promoters capable of transcribing said sequenceto RNA or dsRNA upon binding of an appropriate transcription factor tosaid promoters, and c) introducing said vector into the plant.

[0015] Thus, advantageously, the method according to the inventionprovides a particularly selective mechanism for alleviating pestinfestation, and in some cases parasitic infestation of plants, suchthat when the pest feeds on the plant it will digest the expressed dsRNAin the plant thus inhibiting the expression of the DNA within the pestwhich is critical for its growth, survival, proliferation orreproduction. In a preferred embodiment, the pest may be any ofTylenchulus ssp. Radopholus ssp., Rhadinaphelenchus ssp., Heteroderassp., Rotylenchulus ssp., Pratylenchus ssp., Belonolaimus ssp., Canjanusssp., Meloidogyne ssp., Globodera ssp., Nacobbus ssp., Ditylenchus ssp.,Aphelenchoides ssp., Hirschmenniella ssp., Anguina ssp., Hoplolaimusssp., Heliotylenchus ssp., Criconemellassp., Xiphinemassp., Longidorusssp., Trichodorus ssp., Paratrichodorus ssp., Aphelenchs ssp. The DNAsequence or fragment thereof according to this aspect of the inventionmay be cloned between two tissue specific promoters, such as two rootspecific promoters.

[0016] A further aspect of the invention concerns the vector used ineach of the methods of the invention for constructing said library,which vector comprises two identical promoters oriented such that theyare capable of initiating transcription of DNA sequence located betweensaid promoters to dsRNA upon binding of an appropriate transcriptionfactor to said promoters. The DNA sequence may, for example, include amultiple cloning site. Preferably, the expression vector comprises anucleotide sequence encoding a selectable marker. In one embodiment thenucleotide sequence encoding said selectable marker is located betweentwo identical promoters oriented such that they are capable ofinitiating transcription of DNA located between said promoters to doublestranded RNA upon binding of an appropriate transcription factor to saidpromoters. Preferably, the selectable marker comprises a nucleotidesequence encoding sup-35, for introduction into C. elegans having apha-1 mutation.

[0017] Preferably, the transcription factor comprises either a phagepolymerase which binds to its corresponding promoter or a C. elegansspecific promoter and even more preferably T7 polymerase. Preferably,the vector includes a multiple cloning site between said identicalpromoters.

[0018] In a further aspect of the invention there is provided anexpression vector for expressing an appropriate transcription factor foruse in a method according to the invention which vector comprises asequence of nucleotides encoding said transcription factor operablylinked to suitable expression control sequences. Preferably, theexpression control sequences include promoters which are inducible,constitutive, general or tissue specific promoters, or combinationsthereof. Preferably, the transcription factor comprises a phagepolymerase, and preferably T7, T3 or SP6, RNA polymerase.

[0019] A further aspect of the invention provides a selection system foridentifying transformation of a cell or organism with a vector accordingto the invention which system comprises a vector according to theinvention wherein said selectable marker comprises a nucleotide sequencecapable of inhibiting or preventing expression of a gene in said cell ororganism which gene is responsible for conferring a known phenotype.Preferably said nucleotide sequence corresponds to a part of or isidentical to said gene conferring said known phenotype, and whichnucleotide sequence is itself located between two identical promoterscapable of initiating transcription of double stranded RNA upon bindingof an appropriate transcription factor thereto. Alternatively, thenucleotide sequence comprises a nucleotide sequence which is a part ofor identical to said gene sequence which confers a known phenotype onsaid cell or organism, and which is such that following integration ofsaid vector by homologous recombination in the chromosome of said cellor organism said sequence inhibits expression of said gene sequenceconferring said known phenotype. Preferably, according to thisembodiment the nucleotide sequence comprises stop codons sufficient toprevent translation of the-nucleotide sequence following integrationinto said chromosome. Preferably, the known gene sequence comprises asup-35 gene or a fragment thereof which is selectable by identifyingoffspring growing at a temperature above 25° C. following introductionin a pha-1 et123ts mutant C. elegans worm.

[0020] In a further aspect of the invention provides said known genesequence comprises a sup-35 gene or a fragment thereof which isselectable by identifying offspring growing at a temperature above 25°C. following introduction of said vector in a pha-1 et123ts mutant C.elegans worm. An even further aspect comprises a method of assigningfunction to a DNA sequence of a multicellular organism which methodcomprises a) providing i) a construct comprising said DNA fragmentcloned between two promoters capable of promoting transcription in saidmulticellular organism, in a multicellular organism capable ofinitiating transcription from said promoter; b) identifying thephenotype of said multicellular organism compared to wild type.

[0021] The present invention may be more clearly understood by thefollowing examples which are purely exemplary with reference to theaccompanying figures, wherein:

[0022]FIG. 1 is a nucleotide sequence of plasmid PGN1 in accordance withthe present invention.

[0023]FIG. 2 is a nucleotide sequence of plasmid PGN100 in accordancewith the present invention.

[0024]FIG. 3 is a schematic representation of the vectors used and thetransformation regime used in the methods according to the presentinvention.

[0025]FIG. 4 is an illustration of an expression vector used inaccordance with the invention.

[0026]FIG. 5 is a schematic illustration of the T7 RNA polymeraseexpression vectors used for transforming C. elegans.

[0027]FIG. 6 is an illustration of plasmid PGN1.

[0028]FIG. 7 is a diagrammatic representation of an enhanced vector fordsRNA inhibition encoding sup-35 dsRNA.

[0029]FIG. 8 is an illustration of a vector for integration into thegenome of C. elegans.

[0030]FIG. 9 is an illustration of the position of a DNA sequence(s)relative to a suitable promoter to initiate expression of dsRNA from theDNA sequence(s).

[0031]FIG. 10 is a representation of plasmid pGN108.

[0032]FIG. 11 is a representation of plasmid pGN105.

[0033]FIG. 12 is a representation of plasmid pGN400.

[0034]FIG. 13 is a representation of plasmid pGN401.

[0035]FIG. 14 is a representation of plasmid pGN110.

[0036]FIG. 15 is a representation of plasmid pAS2 with forward andreverse T7/T3/SP6 promoters.

[0037]FIG. 16 is a representation of plasmid pGAD424 with forward andreverse T7/T3/SP6 promoters.

[0038]FIG. 17 is a representation of plasmid pAS2-cyh2-HA+, bothT7-final.

[0039]FIG. 18 is a representation of plasmidpGAD424-without-FULL-ICE-BOTH-T7.

[0040]FIG. 19 (a) is a representation of plasmid pGN205 and(b) is arepresentation of plasmid pGN207.

Example A Construction of an Ordered and Hierarchical Pooled cDNALibrary and Applications Thereof

[0041] A Random Ordered and Pooled Library:

[0042] The vector is an E. coli vector harboring two T7 promoters, witha multiple cloning site (MCS) in between. The two promoters areorientated towards each other, and towards the MCS. In the presence ofT7 RNA polymerase, expressed in E. coli, C. elegans or any otherorganism, RNA will be produced, starting from the two T7 promoters. Asthese are oriented in the opposite sense, both strands of RNA will beproduced from the DNA inserted (cloned) into the MCS in between the twopromoters which results in the generation of double stranded RNA (dsRNA)upon binding of the T7 RNA polymerase thereto.

[0043] A C. elegans cDNA library is constructed in the MCS usingstandard molecular biological techniques. The library is transformedinto E. coli and the resulting E. coli are grown in culture and storedin 96 multi-well plates. At this stage, plasmid DNA can be isolated andstored in 96-multi-well plates corresponding to those of the E. colicolonies. Approximately 100,000 colonies are scored. In this way, thelibrary will harbor approximately 5 times the total expressed cDNAvariation of C. elegans, which gives the opportunity for low expressedsequences to be present in the library. This will result inapproximately 1041 96-well plates. The plates are hierarchical pooled asnecessary. For the present pooling of the clones is arranged in a rangeof 10 to 100. If the hierarchical pooling is per 8 or 12 (numbers aremore convenient: as 96-well plates have a 8 to 12 grid), this willresult in approximately 87 multi-well plates and approximately 8352wells. If hierarchical pooling is per 96 wells, which is a full plate,this results in approximately 11 plates and approximately 1041 wells. Atany stage of the hierarchical pooling, plasmid DNA can be isolated,which would be less elaborate as less plates are used, but will resultin a loss of complexity although this should not be the case in thepooling per 12. The pooling of the DNA can also be carried out with theoriginal DNA.

[0044] The experiments below describe how the hierarchical poolingshould be performed, both for the DNA and for the E. coli library.

An Ordered Library for RNAi Technology, Harboring Every Gene of the C.elegans Genome, with Applications Thereof

[0045] As the genome-sequencing project is coming to an end, thisinformation can be used in the application of T7 RNA inhibitiontechnology. Every gene of the C. elegans genome can be cloned using PCRtechnology. In preference, exons will be cloned with a minimal length of500 bp. If the exons are too small, smaller fragments will be isolatedwith PCR, or even parts of introns and neighboring exons will beisolated with PCR technology so that at least a sufficient part of thetranslated region of the gene is cloned. For this, at least 17000 PCRreactions need to be performed. This collection of PCR products will becloned in a T7 vector as described (two T7 promoters oriented towardseach other with a multiple cloning site in between). Every PCR productis cloned independently, or can be used to generate a random library,analogous to the described cDNA library. If every PCR product is clonedindividually, the resulting bacteria and plasmid DNA can be pooled invarious ways. Firstly, this collection of individually cloned PCRproducts in the T7 RNAi vector can be pooled randomly, as described inthe random library. This pooling can also be done in a more rationalway. For instance, the genes of the C. elegans genome can be analyzedusing bioinformatic tools (in silico biology). Various genes of thegenome will belong to a gene family, or will have homologues in thegenome. These members of the gene family will be pooled, or the members,being homologues will be pooled. In this way the total number of about17000 clones is reduced to a more useable quantity. This library can beused to screen for phenotypes in the methods according to the invention.The resulting phenotype gives a functional description to the gene orgene family or gene homologues of the C. elegans genome. As the libraryconsists of a part of every gene in the genome, this method enablesdescription of the full genome in functional-phenotypic terms. For thisthe double stranded RNA (dsRNA) needs to be introduced in the worm. Thisintroduction of clones alone, or pooled clones, being random pooling orrational pooling can be achieved in several ways as described.

Example of a Vector for the Expression of Double Stranded RNAi

[0046] Any vector containing a T7 promoter may be used, and whichcontains a multiple cloning site (there are many commerciallyavailable). Primers containing the T7 promoter and a primer with thereverse complementary strand, both with the appropriate ends aredesigned. These primers can be hybridized, and if well designed, clonedin the vector of choice. The minimal sequence for a T7 promoter isTAATACGACTCACTATAGGGCGA. Although any vector can be used for theconstruction of a T7 expression vector there follows an example of howto achieve this with the vector pGEM-3zf(−).

[0047] Vector pGEM-3zf(+) (PROMEGA) was digested with HindIII and SalI

[0048] Primers oGN1 and oGN2 were mixed together at a finalconcentration of 1 μg/30 μl boiled and cooled slowly to roomtemperature.

[0049] The primer was ligated into the vector using standard ligationprocedures. The resulting vector is pGN1 (shown in FIG. 1) and containstwo T7 promoters oriented towards each other, and harbors a multiplecloning site in between.

[0050] Sequences of oGN1 and oGN2 are:

[0051] oGN1: AGC TGT AAT ACG ACT CAC TAT AGG GCG AGA AGC TT

[0052] oGN2: TCG AAA GCT TCT CGC ATA ATA GTG AGT CGT ATT AC

Example of the Construction of a Library

[0053] RNA may be isolated from every organism that is sensitive toRNAi. In general the isolated RNA is then copied into double strandedcDNA, and subsequently prepared in suitable vectors for cloning. Severalprocedures exist and molecular biology kits can be purchased fromvarious firms including promega, clontech, boehringer Mannheim, BRL, etcwhich enable:

[0054] isolation of RNA,

[0055] eventually polyA RNA can be isolated (several techniques and kitsavailable)

[0056] first strand synthesis with AMV reverse transcriptase, randomhexameric primers and/or oligo (dT) primer

[0057] second strand synthesis with Rnase H, DNA PolymeraseI,

[0058] flush ends with T4 DNA Polymerase

[0059] addition of an adaptor with T4 DNA ligase.

[0060] eventually treatment with T4 polynucleotide Kinase

[0061] cloning of the cDNA into the vector.

[0062] The resulting ligation mixture can be considered as the cDNAlibrary. The ligation contains all cDNA of the procedure ligated intothe vector of interest. To order the library, the ligation needs to betransformed into E. coli strains.

Application of this E. coli or DNA Library

[0063] T7 RNA producing strain:

[0064] a standard strain is BL21 (DE3): F-ompT[lon]hsds(r- m-; and E.coli B strain) λ (DE3). Eventually variants-of PL21 (DE3) can be used,although BL21 (DE3)pLysS is used.

[0065] any other E. coli strain which produces the T7 RNA polymerase,which may be available needs to be constructed. This can be generatedeasily using a phage, which is commercially available, in this case, theλCE6 vector (provided by Promega) is used. Almost every E. coli straincan be transfected with this phage and will produce T7 RTA polymerase.

[0066] a RNAseIII mutant E. coli:

[0067] Various strains are in principle available, we chose in a firstexperiment to use strain AB301-105: rna-19, suc-11, bio-3, gdhA2, his95,rnc-105, relA1, spoT1, metB1. (Kinder et al. 1973 Mol. Gen. Genet126:53), but other strains may suit better. This strain is infected withλCE6 and so a T7 producing variant will be constructed.

[0068] Wild type C. elegans worms can be grown on the bacteria pools.The bacteria is expressing the T7 RNA polymerase. This results in largequantities of dsRNA in the gut of the C. elegans, which will diffuse inthe organism and results in the inhibition of expression. This librarycan now be used for the screening of several phenotypes. This techniquehas the advantage that it is a much faster to detect relevant genes incertain pathways, than the known C. elegans technology. Moreover, if aninteresting phenotype is found, the responsible gene can be clonedeasily.

[0069] Using the hierarchical pooling one can easily find in a secondscreen the relevant clone of the pool. The inserted DNA of this clonecan then be sequenced. This experiment results in genetic andbiochemical DATA in one step.

[0070] Wild type C. elegans strains can be combined with compounds toscreen for phenotype, drug resistance and or drug sensibility. The C.elegans strain can be a mutant strain, screening for an enhancedphenotype, reduced phenotype, or a new phenotype. The C. elegans straincan be a mutant strain, and the library screen can be combined withcompounds. So one can screen for drug resistance, drug sensibility,enhanced phenotype, reduced phenotype, or a new phenotype. The E. colistrain may be any T7 RNA polymerase expressing strain, like BL21 (DE3),for example, but the formation of double strand RNA may be enhanced byusing a special E. coli strain that is RNAseIII negative. RNAseIIIrecognizes specific loops in dsRNA. Eventually, an E. coli strain can beused that is deleted in RNAses other than RNAseIII or an E. coli can beused that is deleted in one or more RNAses. The expression of the T7 RNApolymerase in most known E. coli strains and constructs which areavailable to generate T7 RNA polymerase producing E. coli strains,generally comprise an inducible promoter. In this way the production ofthe T7 RNA polymerase is regulated, and thus the production of thedsRNA. Advantageously, this feature can be used to “Pulse” feed the C.elegans worms at specific stages of growth. The worms are grown on thenon-induced E. coli strains. When the worm has reached the stage ofinterest, the T7 RNA production in the bacteria is induced. This allowsthe studying of the function of any gene at any point in the life cycleof the animal.

Screening the Library for Homologues of Putative Interesting HumanGenes, and Assign Function to These Genes

[0071] Hundreds of genes have been isolated in various projects, beinggenomic projects, differential expressed arrays, hybridization studies,etc. The described cDNA library can provide a way to validate and orassign function to these genes in a fast and efficient manner. First ofall the worm homologue or homologues or the genes need to be identifiedby bioinformatic tools (in silico biology). PCR primers are developedand the cDNA fragment is isolated using PCR technology. PCR can beperformed on the hierarchical pools. The positive pool or individualwells harboring the bacteria that has the appropriate cDNA is fed to C.elegans and the phenotype is scored.

[0072] PCR can be performed on cDNA isolated from C. elegans. Theresulting DNA can be cloned in the T7 vector and transformed in thedsRNA producing E. coli on which the C. elegans worms are then fed.Depending on which way is faster and more reliable a choice needs to bemade.

[0073] If the gene belongs to a gene family, the worm may need to be fedon a mixture of bacteria. Each of them harboring a part of the member ofthe gene family. E. coli strains, growth conditions, combinations withcompounds can be performed as described above.

[0074] If the library rational is used, in which all the genes of C.elegans are cloned in a organized and structured way, the C. eleganshomologue and eventually the other homologues, orthologues, and membersof the gene family can be traced back easily in the library using asilico biology. No PCR is involved in this step, and the bacteria and orDNA can be isolated on which the worm will be grown.

EXAMPLES

[0075] The idea of the series of experiments was to test both the RNAivector and the various E. coli strains that were constructed.

[0076] 1) Construction of a Test Plasmid

[0077] Any cDNA that gives a clear phenotype in the worm whenknocked-out, or used in a RNAi experiment can be used. It is known thatunc-22 is a good candidate, but may other genes are possible. We optedfor a sensitive system that can be used at a later stage. The system wastested with sup-35 in a pha-1 background. Exon 5 of the sup-35 wasisolated by PCR and cloned in the T7 promoter vector pGN1. The resultingvector was designated pGN2. pha-1 (e2123) mutant worms cannot produceoffspring at temperatures higher than 25° C. This is due to adevelopmental problem in embryogenesis. When sup-35 is knocked-out, orinhibited in this strain, offspring may grow at this temperature.Combination of pha-1 mutant worms and sup-35 RNAi is a good system tovalidate the various options.

[0078] 2) Testing the RNAi Using an E. coli Strain that Produces dsRNA.

[0079] pGN2 was introduced in E. coli strain BL21(DE3) and T7 RNApolymerase was induced with IPTG. C. elegans worms (pha-1 (e2123)) wereinoculated on this bacteria, and grown at the restricted temperature of25° C. As this mutant is an embryonic mutant at this temperature, nooffspring will be observed. If the sup-35 gene is efficiently inhibitedby the dsRNA present in the E. coli, offspring will be observed.

[0080] pGN2 was introduced in E. coli strain AB301-105(DE3) and T7 RNApolymerase was induced with IPTG. C. elegans worms (pha-1 (e2123)) wereinoculated on this bacteria, and grown at the restricted temperature of25° C. As this mutant is an embryonic mutant at this temperature, nooffspring will be observed. If the sup-35 gene is efficiently inhibitedby the dsRNA present in the E. coli, offspring will be observed.

[0081] 3) Improving the Worm Strain for Better Uptake of dsRNA.

[0082] Before plating the pha-1 C. elegans on the E. coli strain thatproduce the double stranded sup-35 RNA. The worm was mutagenised withEMS (Methane sulfonic Acid Ethyl). The offspring of this mutagenisedworm is then plated on the bacteria. The worm that feed on this bacteriagive larger offspring which has a mutation that results in animprovement of dsRNA uptake, and can be used for further experiments.

Stable Integration of the dsRNA Producing Vector into the Genome of theT7 RNA Polymerase Producing Worm

[0083] An E. coli vector can be constructed harboring the followingfeatures; Two T7 promoters directed towards each other, with arestriction site or a multiple cloning site in between. Furthermore, thevector may contain the C. elegans sup35 genomic DNA, engineered in sucha way that it contains several stopcodons at various intervals, so thatno full length protein can be expressed form the sup35 genomic DNAfragment as illustrated in FIG. 8. Any cDNA or cDNA fragment can becloned in the multiple cloning site between the two T7 promoters. Whenthis vector is introduced in a C. elegans strain which expresses T7 RNApolymerase, the cDNA or DNA fragment cloned between the two T7 promoterswill be transcribed, generating dsRNA from the cloned fragment.

[0084] The vector is designed to be used in pha-1 (e2123) mutant wormsexpressing T7 RNA polymerase. The expression of the T7 RNA polymerasemay be constitutive or regulated, general or tissue specific. Thesepha-1 (e2123) worms cannot produce offspring at temperatures higher than25° C., which is due to a development problem in embryogenesis. Whensup-35 is inhibited or knocked-out in this stain, offspring may grow atthis temperature.

[0085] When the vector is introduced in the worm, the vector mayintegrate by homologous recombination (Campbell-like integration). Ithas been shown that homologous recombination occurs in C. elegans,although at low frequencies (Plasterk and Groenen, EMBO J. 11:28?-290,1992). Homologous recombination at the sup35 gene will result in aknock-out of the gene as the two resulting sup-35 genes will harbor thestopcodons. The resulting worm, and its offspring, if this recombinationhappens in the eggs, will have a copy of the vector integrated in thegenome. This can be selected as only the worms for which the sup-35 hasbeen knocked-out will have offspring at temperatures higher than 25° C.Furthermore, the resulting worm will stably produce double stranded RNAfrom the DNA fragment cloned between the two T7 promoters. This worm cannow be considered as a stable transgenic worm strain with a reduction offunction of the gene, from which a fragment has been cloned between thetwo T7 promoters.

[0086] The DNA may be delivered to the worm by several techniques,including injection, ballistic transformation, soaking in the DNAsolution, feeding with bacteria. New and other methods that increase thetransformation efficiencies can be considered.

[0087] The target C. elegans strain may in addition, have othermutations than the-pha-1 (e2123) mutation, and may express other genesthan T7 RNA polymerase.

Example B A Yeast Two-hybrid-RNAi Vector

[0088] A yeast two hybrid vector can be constructed harboring the two T7promoters. The vectors can be designed to replicate both in yeast and inE. coil. In general cDNA libraries for the yeast two hybrid system aremade in the Gal4 or LexA vectors. The library is constructed in vectorshaving the activation domain of one of these genes. A vector can beconstructed that can still perform in the yeast two hybrid screen butwhich also contains two T7 promoters orientated towards each other, witha cloning site therein between. The order of the sequences in theplasmid will then be “plasmid backbone, (GAL4-T7), MCS, T7, backbone”. AC. elegans cDNA library constructed in this vector can be used as astandard yeast two hybrid library in an experiment to isolateinteracting proteins with a given protein. Once a clone is isolated, theplasmid can be introduced in an E. coil strain expressing the T7 RNApolymerase, and hence will produce dsRNA of the cloned fragment. Thebacteria producing this dsRNA can be fed to the worm and phenotypes canbe scored. As in the previous example, this validation procedure for anewly isolated yeast two hybrid clone is remarkably shorter than thestandard procedure, which requires PCR and/or cloning steps, RNAexperiments and/or knock-out experiments. In most cases isolated clonesare sequenced first, and on the basis of the sequence, a decision ismade to continue with further experiments. In the present inventionevery isolated clone can easily be introduced into the appropriate E.coli and fed to the worm. Validation is then performed by phenotypeanalysis.

[0089] To apply this procedure a yeast two hybrid was performed using aknown gene as bait and the newly constructed library as the target.Proteins coded by the clones in the target that interact with the baitprotein, will result in positive yeast clones expressing the reportermolecule such as can be observed by LacZ staining with X-gal. Theplasmid coding for the target protein is isolated directly from theyeast strain and introduced in E. coli. The E. coli is T7 RNA polymeraseproducing E. coli. In this case, double stranded RNA is produced fromthe DNA cloned in the multiple cloning site of the vector. When thisdsRNA is fed to the worm using the methods described previously, thegene has inhibited in the worm, resulting in a particular phenotype.

[0090] This yeast two hybrid vector can advantageously be used toconstruct an ordered and hierarchically pooled library as described inthe previous example.

[0091] A yeast strain can also be constructed that conditionallyproduces T7 RNA polymerase. After yeast two hybrid experiments, theexpression of the T7 polymerase could be induced, resulting in theproduction of dsRNA in the yeast cell. Consequently the yeast could befed to the worm. Evidence is available showing that the C. elegans wormscan feed on yeast.

Construction of a T7 RNA Polymerase Producing Strain, and ApplicationsThereof

[0092] A C. elegans strain can be constructed that expresses T7 RNApolymerase. The expression can be general and constitutive, but couldalso be regulated under a tissue specific promoter, an induciblepromoter, or a temporal promoter or a promoter that harbors one of thesecharacteristics or combination of characteristics. DNA can be introducedin this C. elegans strain. This is done either by injection, by shootingwith particles, by electroporation or as aforementioned by feeding. Ifthe DNA is a plasmid as described in the previous examples, i.e. aplasmid harboring a cloned cDNA fragment or a PCR fragment between twoflanking T7 promoters, then dsRNA of this cDNA or PCR fragment is formedin the cell or whole organism resulting in down regulation of thecorresponding gene. The introduced DNA can have an efficient transientdown regulation. The introduced DNA can form an extrachromosomal array,which array might result in a more catalytic knock-out or reduction offunction phenotype. The plasmid might also integrate into the genome ofthe organism, resulting in the same catalytic knock out or reduction offunction phenotype, but which is stably transmittable.

[0093] Plasmid DNA harboring a cDNA or a part of a cDNA or an EST or anPCR fragment of C. elegans cloned between two T7 promoters as describedin Examples A) and B) can be introduced in the T7 RNA polymerase worm,by standard techniques. Phenotypes can be analysed -DNA from an orderedand pooled library as in Example A) can be introduced in the T7 RNApolymerase worm, by standard techniques (injection, shooting).Phenotypes can be analysed. With the hierarchical pool, the originalclone can be found easily.

[0094] The same procedure can be performed with a mutant worm expressingthe T7 RNA polymerase. Screening for enhanced, reduced or newphenotypes.

[0095] The procedure can be used to enable screening of compounds.Screening with either a wild-type strain or a mutant strain for enhancedor new phenotypes.

[0096] The DNA could be introduced in the worm by new methods. One ofwhich is the delivery of DNA by E. coli. In this case the hierarchicalpooled library is fed to the animal. To prevent digestion of the E. coliDNA in the gut of the nematode, preferentially a DNAse deficient C.elegans will be used, such as nuc-1 (e1392). This procedure would be oneof the most interesting as it would be independent of transformationefficiencies of other techniques, and generally faster and lesslabourious.

[0097] 2) Putative Enhancements of the Method.

[0098] A vector is designed, so that it harbors the sup-35 cDNA or apart of this cDNA, cloned in between two T7 promoters. The rest of thevector is as described in Examples A) and B). This vector can beintroduced into a pha-its mutant C. elegans. A temperature selectionsystem exists in this case and only those worms which have taken up theDNA and express the double stranded sup-35 RNA will survive atrestricted temperatures. The hierarchical pooled library can bedelivered by any method described above.

[0099] The vector can be used to construct a library that is introducedin a T7 RNA polymerase expressing E. coli. In this case we have ananalogous screening as in part A) with an additional screening for wormswhere the dsRNA of sup-35 is active.

[0100] The DNA and or dsRNA of sup-35 could be delivered on a differentplasmid. For the feeding, both DNA feeding (Example C) or dsRNA feedingExample A) and B), this means that the two plasmids could be present inone bacterium, or that the worm is fed on a mixture of bacteria, one ofwhich harbors the sup-35 construct.

Example of the Construction of a T7 RNA Producing C. elegans

[0101] To produce T7 RNA polymerase in the worm, several possibilitiesare possible. The T7 polymerase can be expressed under variouspromoters, being inducible promoters, constitutive promoters, generalpromoters and tissue (cell) specific promoters, or combinations ofthose. Examples of these promoters are the heatshock promoter hsp-16,the gut promoter ges 1, the promoter from cet858, but also the promoterof dpy 7 and the promoter element GATA1. In this example the T7 RNApolymerase is expressed under the control of the hsp-16 promoter that isavailable in the pPD49.78 vector. The T7 RNA polymerase is isolated as aPCR product using the primers of GN3 an GN4.

[0102] The resulting PCR product is digested with NheI and NcoI, as isthe vector in which we want to clone, being the Fire vector pPD49,78.The resulting vector is pGN100 illustrated in FIG. 2 oGN3: CAT GGC AGGATG AAC ACG ATT AAC ATC GC oGN4: ATG GCC CCA TGG TTA CGG GAA CGC GAA GTCCG pGN100 is included.

[0103] The vector is introduced into the worm using standard techniques,such as micro injection, for example.

[0104] The following strains were then constructed:

[0105] Wild-type (pGN100)

[0106] nuc-1 (e1392) (pGN100)

[0107] pha-1 (e2123) (pGN100)

[0108] pha-1; nuc-1 (pGN100)

[0109] All of these strains are able to produce T7 RNA polymerase whentemperature induced or alternatively by metals such as application ofheavy cadmium or mercury. The procedure for temperature induction is toshift the animal to a temperature of 30-33° C. for at least one hour,then the animal can be shifted back to standard temperatures (15-25°C.).

[0110] The wild type strain producing T7 RNA polymerase can be used forthe production of any RNA in the worm. More specifically, the plasmidsfrom the described libraries can be introduced in these worms, andphenotypes can be scored.

[0111] The nuc-1 mutant worm will be used to introduce DNA via bacteriaon which the worm feed. As the nuc-1 worm does not digest the DNA, theplasmid DNA can cross the gut wall. If taken up by the cells thatproduce the T7 RNA polymerase, dsRNA will be produced thus inhibitingthe gene from which the RNA was transcribed.

[0112] The pha-1 mutant strain that produced T7 RNA polymerase can beused to enhance the procedures as described above. DNA can be introducedby shooting, micro injection or feeding. More specifically this straincan be used for the vectors that produce dsRNA from sup-35 and from thegene of interest, the latter can be a PCR product, a cDNA, or a libraryas described.

[0113] The pha-1; nuc-1 mutant producing T7 RNA polymerase can be usedfor the bacterial delivery of the DNA. DNA will preferentially be theplasmid that produce dsRNA from both sup-35 and the gene of interest.The worm strain will preferentially produce the T7 RNA polymerase in thegut. Delivery will preferentially happen by feeding the worm on bacteriaharboring the plasmid.

Application of the RNAi Technology in Plants

[0114] Nematodes are responsible a large part of the damage inflicted onplants and more particularly to plants used in the agriculturalindustry. The RNAi procedures according to the invention can be appliedto plants to prevent these parasitic nematodes from feeding longer. In afirst step, a DNA fragment is isolated from the parasitic plant nematodethat is critical for the animals survival or growth, or to feed or toproliferate. Any gene from which the expression is essential is suitablefor this purpose.

[0115] A part of this gene, an exon or cDNA is cloned. This DNA fragmentcan be cloned under the influence of a tissue specific promoterpreferably a root specific promoter even more preferably between tworoot specific promoters. The DNA of the cloned gene under the control ofthe root specific promoter can be introduced in the plant of interest,using plant transgenic technology. For every parasitic nematode, adifferent piece of DNA may be required and likewise for every plantrace, a different promoter will be needed.

[0116] The root will produce RNA or dsRNA from the introduced piece ofDNA when root specific promoter is utilised. As the nematode feeds onthe plant, the RNA and/or dsRNA will be consumed or ingested by thenematode. The RNA and/or dsRNA can enter the cells of the nematode andperform its inhibitory action on the target DNA. Depending on the natureof the cloned DNA piece of worm, the nematode will not be able tosurvive, to eat, proliferate, etc in any case preventing the animal offeeding longer on the plant, and thus protecting the plant.

Construction of a T7 RNA-polymerase Producing C. elegans

[0117] To produce a T7 RNA polymerase or other RNA polymerases inanimals, and more particularly in nematodes and most particularly in C.elegans, several possibilities can be envisaged. The T7 RNA polymerasecan be expressed under various promoters. These promoters may beinducible promoters, constitutive promoters, general promoters, tissuespecific promoters, or combinations of those.

Example 1 Construction of a Basic Vector for Expression of T7 Polymerasein C. elegans

[0118] The T7 polymerase coding sequence was PCR amplified from_λ CE6(Novagen, Madison, USA) using the primersoGN26(ATGGAATTCTTACGCGAACGCGAAGTCCG) andoGN46(CTCACCGGTAATGAACACGATTAACATCGC), using standard procedures (PCR, Apractical A practical approach, 1993, Ed. J. McPherson, et al, IRLPress). The resulting DNA fragment encoding for the T7 RNA polymerasewas digested with AgeI and EcoRI and inserted into the Fire vectorpPD97.82 digested with AgeI and EcoRI. The resulting construct encodesfor an open reading frame of T7 RNA polymerase in fusion with the SV40large T antigen nuclear localization signal (NLS) with amino acidsequence MTAPKKKRKVPV. This nuclear localization signal sequence isrequired to translocate the T7 RNA polymerase from the cytoplasm to thenucleus, where it is able to bind to its specific promoters, designatedT7 promoters. Upstream of the coding sequence for the T7polymerasefusionprotein is a minimal promoter (myo-2) preceded by a multiple cloningsite (MCS) in-which several C. elegans promoters can be inserted. Thisplasmid (pGN105 shown in FIG. 11) is a basic T7 RNA polymerase plasmidwhich enables the expression of T7polymerase in C. elegans. Derivativesof this plasmid wherein promoters are cloned into the multiple cloningsite, allow for the inducible, constitutive, general and tissue specificexpression of T7 RNA polymerase in C. elegans, as expression will beregulated by the promoter cloned in the multiple cloning site.

[0119] Although not restricted to these examples, for the followingpromoters it is known that they induce expression in the followingtissues.

[0120] let-858 (ubiquitous expression), myo-2 (pharynx expression),myo-3 (body wall muscles) , egl-15 (vulval muscles), unc-119(pan-neuron).

Example 2 Construction of a Vector for Expression of T7 RNA Polymerasein C. elegans Muscle Tissue

[0121] The T7 RNA polymerase coding sequence was PCR amplified from_λCE6 using the primers oGN43 (GCCACCGGTGCGAGCTCATGAACACGATTAACATCGC) andoGN44 (CACTAGTGGGCCCTTACGCGAACGCGAAGTCCG) digested with AgeI/SpeI andinserted in the pGK13 vector digested with AgeI/SpeI. (This vectorcontains the strong SERCA promoter which drives expression in thepharynx, the vulval muscle, the tail and the body wall muscle). Anuclear localization signal (NLS) of SV40 large T antigen was insertedin front of the T7 polymerase coding sequence by insertion of twooverlapping oligo's oGN45 (CCGGATGACTGCTCCAAAGAAGAAGCGTAAGCT) and oGN46(CTCACCGGTAATGAACACGATTAACATCGC) into the SacI/AgeI restriction sites.The resulting construct was called pGN108 as shown in FIG. 10.Introduction of this plasmid into C. elegans results in the expressionof T7 RNA polymerase in the pharynx, vulva muscle, tail and body wallmuscles.

[0122] To test expression and functionality of T7 RNA polymerase in C.elegans under the regulation of the SERCA promoter, pGN108, whichencodes the T7RNA polymerase under the control of the SERCA promoter wasinjected into C. elegans. A test vector was co-injected. This testvector encodes for GFP under the control of a T7 promoter (pGN401 inFIG. 13). The plasmid pGN401 was constructed by inserting twooverlapping oligo's oGN41 (CCCGGGATTAATACGACTCACTATA) and oGN42(CCGGTATAGTGAGTCGTATTAATCCCGGGAGCT) in the SacI/AgeI opened Fire vectorpPD97.82. generating a T7 promoter. Furthermore a selection marker wasco-injected to select for transformants (rol6, pRF4). The latterselection vector pRF4 is well known to person skilled in the art.Transgenic F1 could easy be isolated as they display the rol 6phenotype. These transgenic C. elegans all expressed GFP in the pharynx,the vulval muscle, the tail and the body wall muscle. This data showclearly that the T7 RNA polymerase is functionally expressed under theregulation of the SERCA promoter, and that the expressed T7 RNApolymerase binds to the T7 promoter present in pGN401 and initiatestranscription of the GFP gene, which is then functionally expressed,resulting in fluorescence in the muscle tissues where SERCA is inducingthe expression of the T7 RNA polymerase.

Example 3 Construction of a Vector for Ubiquitous Expression of T7Polymerase in C. elegans

[0123] The NLS-T7 RNA polymerase fusion gene was isolated from pGN108with XmaI/Bsp1201 and cloned into the Fire vector pPD103.05 digestedwith XmaI/Bsp120I. This results in a vector wherein the T7 RNApolymerase is cloned under the regulation of the let858 promoter. Thisspecific promoter enables the expression of T7 RNA polymerase in alltissues. The resulting plasmid was named pGN110 (FIG. 14)

Example 4 Construction of a Vector for T7 RNA Polymerase MediatedExpression of DNA Fragments, Genes, and cDNA's Under the Control of a T7Promoter

[0124] The Fire vector pPD97.82 was digested with SacI/AgeI and a T7promoter sequence was generated by insertion of two overlapping oligo'soGN41 (CCCGGGATTAATACGACTCACTATA) and oGN42(CCGGTATAGTGAGTCGTATTAATCCCGGGAGCT) into the SacI/Age/restrictionendonuclease sites. This construct (pGN400 FIG. 12) contains a GFP openreading frame cloned between SacI and EcoRI restriction endonucleasesites under the regulation of the T7 promoter. Any gene, cDNA, or DNAfragment can be cloned in this vector by deleting the GFP gene as aAgeI/SacI fragment and cloning the DNA fragment of interest into thevector. Preferentially the DNA fragment of interest can be obtained byPCR amplification, inserting the SacI/AfeI sites in the primers. Theresulting DNA fragment after PCR amplification is the digested and theGFP gene in pGN400 is replaced by the amplified DNA fragment. Everyvector that contains a T7 promoter could be used for the purpose of T7RNA polymerase induced expression in C. elegans, such as thecommercially available pGEM vectors and the pBluescript vectors. This isclearly shown by the pGN401 vector which expresses GFP under theregulation of the T7 promoter in a transgenic C. elegans which expressesT7 RNA polymerase.

[0125] The use of pGN400 has the advantage that the vector includes a3′UTR fragment from unc-54 which enhances the transcription or stabilityof the RNA.

Generation of Permanent, Tissue Specific “Pseudo knock-out” RNAi C.elegans Lines

[0126] At present, gene knock outs in C. elegans are obtained afterrandom, large scale mutagenesis and PCR base sib-selection. This methodis bulky, very time consuming and tedious. It has been described thatintroducing double stranded RNA into a cell results in potent andspecific interference of expression of endogenous genes. In C. elegansgene expression can be down regulated by injection of RNA into the bodycavity of the worm, soaking the worm in a solution containing dsRNA orfeeding E. coli that express dsRNA corresponding to the gene ofinterest. C. elegans cells have the ability to take in dsRNA from theirextracellular environment. It has been reported that mRNA is the targetof this ds RNA mediated genetic interference (Montgomery and Fire 1998).It is also suggested that the targeted RNA is degraded in the nucleusbefore translation can occur. Although the RNAi mediated reduction ofgene expression can be passed on to the next generations, heritabilityis poor and the effect is rapidly lost during further offspring. This isprobably due to a continued decrease of the dsRNA pool. We propose herea method to construct C. elegans lines with a permanent, inheritable,RNAi phenotype. The method encompasses the generation of transgenic C.elegans lines by introducing plasmids containing cDNA fragments of thetarget gene in the sense and antisense orientation under control of aworm promoter or by transcription of an inverted repeat of the cDNA froma single construct. Alternatively, ds RNA can be transcribed from avector harboring a cDNA flanked by two T7 promoters in a C. elegansstrain that expresses T7 polymerase. The result is a transgenic wormwith an heritable stable “pseudo knock-out” phenotype. The expression ofthe cDNA or the T7 polymerase can be general and constitutive but couldalso be regulated under a tissue specific promoter. In contrast to RNAiinduced by external ds RNAi (injected, soaked or feeded) this methodwould enable to obtain conditional, tissue specific inhibition of geneexpression.

Inhibition of unc-22 Expression by RNA Interference Results in a“Twitching” Phenotype

[0127] Unc 22 cDNA (exon 22) was cloned in sense and antisenseorientation in pPDl03.05. (A. Fire nr L2865) containing the let 858promoter that is capable of expressing RNA sequences in all tissues. Theresulting plasmids were named pGN205 (FIG. 19a) and pGN207 (FIG. 19 b).These constructs were introduced into C. elegans together with aselectable marker (rol-6; GFP). Transgenic F1 individuals (expressingrol-6 or GFP) showed a “twitching” phenotype indicating that RNAi couldbe mediated by endogenous transcription of RNA from transgenic DNA. TheRNAi phenotype co-segregated with the selectable marker into furtheroffspring. This resulted in the generation of C. elegans lines withpermanent RNAi phenotype.

Generation of Stable Lines T7 RNA Polymerase Lines and Generation ofDual Transgenic Worms

[0128] An expression system in C. elegans based on an exogenous RNApolymerase demands two plasmids. One is encoded for the RNA polymeraseunder the control of a specific promoter, while the other plasmidencodes for the DNA fragment to be expressed, under the regulation ofthe T7 promoter. In the case of semi stable RNAi also designated pseudostable knockouts, the DNA of interest is cloned between two T7 promotersso that dsRNA can be produced.

[0129] As the T7 RNA polymerase expression system is known to be a highexpression system this will result in problems to generate duallytransgenic animals. If the gene to be expressed in the C. elegansnematode is toxic, this will result in lethal effects and hence in theconstruction of a C. elegans without highly regulated stable expressionof the gene of interest. If the gene of interest is essential for thesurvival of the organism, RNAi with a DNA fragment from this gene willalso result in lethal effects, so that pseudo- stable knockouts are notpossible.

[0130] To overcome this problem the present inventors have designed asystem consisting of two transgenic animals. The first animal istransgenic for the T7 RNA polymerase, This T7 RNA polymerase can beexpressed in all cells or specific cells or tissues as has been shown inprevious examples. The second transgenic animal is transgenic for theDNA fragment of interest. This can be a gene or cDNA linked to a T7promoter, or if one wants to perform RNAi a DNA fragment of such genecloned between two T7 promoters.

[0131] Both transgenic animals are viable and do not show any aberrantphenotypes. This is because the T7RNA polymerase expressed in the firsttransgenic organism is not toxic for the organism, even if expressed atrelative high levels. In the second transgenic organism, the gene ofinterest is not expressed or the dsRNA is not produced as thesetransgenic animals do not contain the T7 RNA polymerase.

[0132] Expression of the gene or cDNA of interest or RNAi with a DNAfragment can now be obtained by mating is the two transgenic animals.The offspring of these are dually transgenic and express the gene ofinterest or express dsRNA of the DNA fragment of interest. To generatesufficient males in such a mating, one of the transgenic animals malescan be a C. elegans mutant with a phenotype favouring generation ofmales. An Example of such a mutant is him-5. Preferentially such amutant will be used to make a C. elegans transgenic for T7 RNApolymerase, while the hermaphrodite harbors the DNA fragment under theregulation of the T7 promoter.

[0133] To select efficiently for the dual transgenic offspring a secondtransgene can be introduced in the second transgenic animal. Thistransgene contains a reporter gene under the regulation of the T7promoter. The reporter gene can be GFP, luciferase, Beta galmactosidase,or beta-lactamase. an example of such a ttransgene are the vectorspGN400 and pGN401.

[0134] To obtain inducible, tissue specific expression of a transgene inC. elegans we can make male stock (i.e. him-5) carrying the T7polymerase construct under the control of different C. elegans promotersthat enable tissue specific expression such as). This males can becrossed with hermaphrodites carrying the gene of interest under thecontrol of a T7 promoter.

[0135] Furthermore, the transgenes can be integrated into the genome ofthe animal. Methods to generate stable integration of a plasmid into thegenome of the animal have been described (Methods in cell biology, Vol.48, 1995, ed. by epstein and Shakes, academic press) and involveradiation of the animal. This can be done for both animals animals, butpreferentially, the animals expressing the T7 RNA polymerase are subjectto such traetment. This result in a collection of C. elegans nematodesthat stably express T7 RNA polymerase under the control of variouspromoters. examples of such promoters are the myo-2 (pharynxexpression), myo-3 (body wall muscles), egl-15 (vulval muscles), unc-119(pan-neuron), SERCA (muscles), let858 (all cells) ges-1 (gut).

Construction of RNAi T7 Promoter Yeast Two Hybrid Vectors

[0136] pGAD424 with Forward and Reverse T7/T3 and or Sp6

[0137] In most two-hybrid experiments a cDNA library is cloned inplasmid pGAD424 (FIG. 16) which has been engineered with additionalrestriction sites in the polylinker such as a Ncol site (Clontech). Thislibrary allows for screening of binding proteins in a yeast two hybridexperiment. We constructed a new yeast two hybrid vector with the samepossiblilities to perform yeast two hybrid, but which contain twoaddditional T7 promoters, so that the vector can be used for T7 RNApolymerase induced pseudo-stable knock-outs. For this we inserted aforward T7 by using a T7- linker (consisting of the following primersaattcttaatacgactcactatagggcc and catgggccctatagtgagtcgtattaag) into theEcoRI-Ncol site of pGAD424. The resulting vector was designatedpGAD424-without-FULL-ICE-both-T7. Care was taken to eliminate stopcodons and using maximal polylinker compatible amino acids. We adoptedthe same strategy for the reverse T7 (consisting of both primersgatccgtcgacagatctccctatagtgagtcgtattactgca andgtaatacgactcactatagggagatctgtcgacg) with BamH1 and Pst1. To avoid lossof SalI, we included this site in the primer.

[0138] The SalI site is important as most libraries are cloned in thissite, adapters are available. This makes the newly constructed vectorcompatible with existing vectors.

pAS2 with with Forward and Reverse T7/T3 and or Sp6

[0139] An analogous yeast two hybrid vector was constructed based onpAS2 (Clontech). By partial EcoRV digestion we were able to remove asignificant part of the cyh2 gene. The right construct can be isolatedand checked by a restriction digest with BglII. this restriction site ispresent in the EcoRV fragment of PAS2 to be eliminated. This elimatesthe cyh2 gene which is slightly toxic gene and involved in growthretardation. This gene is non-essential for the performing of RNAi andYeast two hybrid experiments. After the elimination of the EcoRVfragment, The EcoRI restriction site which is located between the DNAsequence encoding for GAL4DB and HA (epitope) becomes unique for theplasmid, and can be used to subsitute HA with a T7 promoter containinglinker. This ensures persistence of all restriction sites, allowing bothin frame cloning and compatibility with previous vectors and pGAD424. Weused the following linker (primers: aattcttaatacgactcactatagggca andtatgccctatagtgagtcgtattaag) using EcorI and Nde1 cloning sites. Weadopted the same strategy for the reverse T7 (primers:gatccgtcgacagatctccctatagtgagtcgtattactgca catgggccctatagtgagtcgtattaagand gtaatacgactcactatagggagatctgtcgacg) with BamH1 and Pst1. To avoidloss of Sal1 we included it in the primer. The resulting vector wasdesignated pAS2-cyh2-HA+both T7-final.

[0140] Having the T7 promoter (or alternatively the T3, or SP6 promoter)in pGAD424 allows to go quickly from interacting protein to RNAi andassigning function to the isolated DNA fragment. An additional advantageis the ability to make by in vitro transcription coupled to in vitrotranslation (There is an ATG in frame with either GAL4DB or GAL4AD)labeled protein which can be used for in vitro controls (e.g. pull downassays) of the actual protein-protein interaction.

[0141] The sequences of the plasmids produced and the SP6 and T3polymerase are identified in the Sequence Listing provided below:

1 29 1 3216 DNA Artificial Sequence Description of ArtificialSequenceplasmid DNA 1 gagtgcacca tatgcggtgt gaaataccgc acagatgcgtaaggagaaaa taccgcatca 60 ggcgaaattg taaacgttaa tattttgtta aaattcgcgttaaatatttg ttaaatcagc 120 tcatttttta accaataggc cgaaatcggc aaaatcccttataaatcaaa agaatagacc 180 gagatagggt tgagtgttgt tccagtttgg aacaagagtccactattaaa gaacgtggac 240 tccaacgtca aagggcgaaa aaccgtctat cagggcgatggcccactacg tgaaccatca 300 cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctctaaatcggaa ccctaaaggg 360 agcccccgat ttagagcttg acggggaaag ccggcgaacgtggcgagaaa ggaagggaag 420 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtagcggtcacgct gcgcgtaacc 480 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgtccattcgcca ttcaggctgc 540 gcaactgttg ggaagggcga tcggtgcggg cctcttcgctattacgccag ctggcgaaag 600 ggggatgtgc tgcaaggcga ttaagttggg taacgccagggttttcccag tcacgacgtt 660 gtaaaacgac ggccagtgaa ttgtaatacg actcactatagggcgaattc gagctcggta 720 cccggggatc ctctagagtc gaaagcttct cgccctatagtgagtcgtat tacagcttga 780 gtattctata gtgtcaccta aatagcttgg cgtaatcatggtcatagctg tttcctgtgt 840 gaaattgtta tccgctcaca attccacaca acatacgagccggaagcata aagtgtaaag 900 cctggggtgc ctaatgagtg agctaactca cattaattgcgttgcgctca ctgcccgctt 960 tccagtcggg aaacctgtcg tgccagctgc attaatgaatcggccaacgc gcggggagag 1020 gcggtttgcg tattgggcgc tcttccgctt cctcgctcactgactcgctg cgctcggtcg 1080 ttcggctgcg gcgagcggta tcagctcact caaaggcggtaatacggtta tccacagaat 1140 caggggataa cgcaggaaag aacatgtgag caaaaggccagcaaaaggcc aggaaccgta 1200 aaaaggccgc gttgctggcg tttttcgata ggctccgcccccctgacgag catcacaaaa 1260 atcgacgctc aagtcagagg tggcgaaacc cgacaggactataaagatac caggcgtttc 1320 cccctggaag ctccctcgtg cgctctcctg ttccgaccctgccgcttacc ggatacctgt 1380 ccgcctttct cccttcggga agcgtggcgc tttctcatagctcacgctgt aggtatctca 1440 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgcacgaacccccc gttcagcccg 1500 accgctgcgc cttatccggt aactatcgtc ttgagtccaacccggtaaga cacgacttat 1560 cgccactggc agcagccact ggtaacagga ttagcagagcgaggtatgta ggcggtgcta 1620 cagagttctt gaagtggtgg cctaactacg gctacactagaaggacagta tttggtatct 1680 gcgctctgct gaagccagtt accttcggaa aaagagttggtagctcttga tccggcaaac 1740 aaaccaccgc tggtagcggt ggtttttttg tttgcaagcagcagattacg cgcagaaaaa 1800 aaggatctca agaagatcct ttgatctttt ctacggggtctgacgctcag tggaacgaaa 1860 actcacgtta agggattttg gtcatgagat tatcaaaaaggatcttcacc tagatccttt 1920 taaattaaaa atgaagtttt aaatcaatct aaagtatatatgagtaaact tggtctgaca 1980 gttaccaatg cttaatcagt gaggcaccta tctcagcgatctgtctattt cgttcatcca 2040 tagttgcctg actccccgtc gtgtagataa ctacgatacgggagggctta ccatctggcc 2100 ccagtgctgc aatgataccg cgagacccac gctcaccggctccagattta tcagcaataa 2160 accagccagc cggaagggcc gagcgcagaa gtggtcctgcaactttatcc gcctccatcc 2220 agtctattaa ttgttgccgg gaagctagag taagtagttcgccagttaat agtttgcgca 2280 acgttgttgg cattgctaca ggcatcgtgg tgtcacgctcgtcgtttggt atggcttcat 2340 tcagctccgg ttcccaacga tcaaggcgag ttacatgatcccccatgttg tgcaaaaaag 2400 cggttagctc cttcggtcct ccgatcgttg tcagaagtaagttggccgca gtgttatcac 2460 tcatggttat ggcagcactg cataattctc ttactgtcatgccatccgta agatgctttt 2520 ctgtgactgg tgagtactca accaagtcat tctgagaataccgcgcccgg cgaccgagtt 2580 gctcttgccc ggcgtcaata cgggataata gtgtatgacatagcagaact ttaaaagtgc 2640 tcatcattgg aaaacgttct tcggggcgaa aactctcaaggatcttaccg ctgttgagat 2700 ccagttcgat gtaacccact cgtgcaccca actgatcttcagcatctttt actttcacca 2760 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgcaaaaaaggga ataagggcga 2820 cacggaaatg ttgaatactc atactcttcc tttttcaatattattgaagc atttatcagg 2880 gttattgtct catgagcgga tacatatttg aatgtatttagaaaaataaa caaatagggg 2940 ttccgcgcac atttccccga aaagtgccac ctgacgtctaagaaaccatt attatcatga 3000 cattaaccta taaaaatagg cgtatcacga ggccctttcgtctcgcgcgt ttcggtgatg 3060 acggtgaaaa cctctgacac atgcagctcc cggagacggtcacagcttgt ctgtaagcgg 3120 atgccgggag cagacaagcc cgtcagggcg cgtcagcgggtgttggcggg tgtcggggct 3180 ggcttaacta tgcggcatca gagcagattg tactga 32162 6460 DNA Artificial Sequence Description of Artificial SequenceplasmidDNA 2 ctagcatgaa cacgattaac atcgctaaga acgacttctc tgacatcgaa ctggctgcta60 tcccgttcaa cactctggct gaccattacg gtgagcgttt agctcgcgaa cagttggccc 120ttgagcatga gtcttacgag atgggtgaag cacgcttccg caagatgttt gagcgtcaac 180ttaaagctgg tgaggttgcg gataacgctg ccgccaagcc tctcatcact accctactcc 240ctaagatgat tgcacgcatc aacgactggt ttgaggaagt gaaagctaag cgcggcaagc 300gcccgacagc cttccagttc ctgcaagaaa tcaagccgga agccgtagcg tacatcacca 360ttaagaccac tctggcttgc ctaaccagtg ctgacaatac aaccgttcag gctgtagcaa 420gcgcaatcgg tcgggccatt gaggacgagg ctcgcttcgg tcgtatccgt gaccttgaag 480ctaagcactt caagaaaaac gttgaggaac aactcaacaa gcgcgtaggg cacgtctaca 540agaaagcatt tatgcaagtt gtcgaggctg acatgctctc taagggtcta ctcggtggcg 600aggcgtggtc ttcgtggcat aaggaagact ctattcatgt aggagtacgc tgcatcgaga 660tgctcattga gtcaaccgga atggttagct tacaccgcca aaatgctggc gtagtaggtc 720aagactctga gactatcgaa ctcgcacctg aatacgctga ggctatcgca acccgtgcag 780gtgcgctggc tggcatctct ccgatgttcc aaccttgcgt agttcctcct aagccgtgga 840ctggcattac tggtggtggc tattgggcta acggtcgtcg tcctctggcg ctggtgcgta 900ctcacagtaa gaaagcactg atgcgctacg aagacgttta catgcctgag gtgtacaaag 960cgattaacat tgcgcaaaac accgcatgga aaatcaacaa gaaagtccta gcggtcgcca 1020acgtaatcac caagtggaag cattgtccgg tcgaggacat ccctgcgatt gagcgtgaag 1080aactcccgat gaaaccggaa gacatcgaca tgaatcctga ggctctcacc gcgtggaaac 1140gtgctgccgc tgctgtgtac cgcaaggaca gggctcgcaa gtctcgccgt atcagccttg 1200agttcatgct tgagcaagcc aataagtttg ctaaccataa ggccatctgg ttcccttaca 1260acatggactg gcgcggtcgt gtttacgccg tgtcaatgtt caacccgcaa ggtaacgata 1320tgaccaaagg actgcttacg ctggcgaaag gtaaaccaat cggtaaggaa ggttactact 1380ggctgaaaat ccacggtgca aactgtgcgg gtgtcgataa ggttccgttc cctgagcgca 1440tcaagttcat tgaggaaaac cacgagaaca tcatggcttg cgctaagtct ccactggaga 1500acacttggtg ggctgagcaa gattctccgt tctgcttcct tgcgttctgc tttgagtacg 1560ctggggtaca gcaccacggc ctgagctata actgctccct tccgctggcg tttgacgggt 1620cttgctctgg catccagcac ttctccgcga tgctccgaga tgaggtaggt ggtcgcgcgg 1680ttaacttgct tcctagtgag accgttcagg acatctacgg gattgttgct aagaaagtca 1740acgagattct acaagcagac gcaatcaatg ggaccgataa cgaagtagtt accgtgaccg 1800atgagaacac tggtgaaatc tctgagaaag tcaagctggg cactaaggca ctggctggtc 1860aatggctggc tcacggtgtt actcgcagtg tgactaagcg ttcagtcatg acgctggctt 1920acgggtccaa agagttcggc ttccgtcaac aagtgctgga agataccatt cagccagcta 1980ttgattccgg caagggtccg atgttcactc agccgaatca ggctgctgga tacatggcta 2040agctgatttg ggaatctgtg agcgtgacgg tggtagctgc ggttgaagca atgaactggc 2100ttaagtctgc tgctaagctg ctggctgctg aggtcaaaga taagaagact ggagagattc 2160ttcgcaagcg ttgcgctgtg cattgggtaa ctcctgatgg tttccctgtg tggcaggaat 2220acaagaagcc tattcagacg cgcttgaacc tgatgttcct cggtcagttc cgcttacagc 2280ctaccattaa caccaacaaa gatagcgaga ttgatgcaca caaacaggag tctggtatcg 2340ctcctaactt tgtacacagc caagacggta gccaccttcg taagactgta gtgtgggcac 2400acgagaagta cggaatcgaa tcttttgcac tgattcacga ctccttcggt accattccgg 2460ctgacgctgc gaacctgttc aaagcagtgc gcgaaactat ggttgacaca tatgagtctt 2520gtgatgtact ggctgatttc tacgaccagt tcgctgacca gttgcacgag tctcaattgg 2580acaaaatgcc agcacttccg gctaaaggta acttgaacct ccgtgacatc ttagagtcgg 2640acttcgcgtt cgcgtaacca tggtattgat atctgagctc cgcatcggcc gctgtcatca 2700gatcgccatc tcgcgcccgt gcctctgact tctaagtcca attactcttc aacatcccta 2760catgctcttt ctccctgtgc tcccaccccc tatttttgtt attatcaaaa aaacttcttc 2820ttaatttctt tgttttttag cttcttttaa gtcacctcta acaatgaaat tgtgtagatt 2880caaaaataga attaattcgt aataaaaagt cgaaaaaaat tgtgctccct ccccccatta 2940ataataattc tatcccaaaa tctacacaat gttctgtgta cacttcttat gtttttttta 3000cttctgataa attttttttg aaacatcata gaaaaaaccg cacacaaaat accttatcat 3060atgttacgtt tcagtttatg accgcaattt ttatttcttc gcacgtctgg gcctctcatg 3120acgtcaaatc atgctcatcg tgaaaaagtt ttggagtatt tttggaattt ttcaatcaag 3180tgaaagttta tgaaattaat tttcctgctt ttgctttttg ggggtttccc ctattgtttg 3240tcaagagttt cgaggacggc gtttttcttg ctaaaatcac aagtattgat gagcacgatg 3300caagaaagat cggaagaagg tttgggtttg aggctcagtg gaaggtgagt agaagttgat 3360aatttgaaag tggagtagtg tctatggggt ttttgcctta aatgacagaa tacattccca 3420atataccaaa cataactgtt tcctactagt cggccgtacg ggccctttcg tctcgcgcgt 3480ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 3540ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 3600tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 3660cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg gccttaaggg 3720cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 3780aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 3840ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 3900aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 3960ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 4020gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 4080ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 4140ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 4200gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 4260aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 4320gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 4380aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 4440caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 4500tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 4560acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 4620gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 4680agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 4740gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 4800ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 4860taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 4920agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 4980aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5040ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 5100gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 5160aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 5220aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 5280gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga 5340aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 5400aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 5460cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 5520cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 5580tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 5640tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 5700ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 5760atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 5820tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 5880gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 5940cgccaagctt gcatgcctgc aggtcgactc tagaggatca agagcatttg aatcagaata 6000tggagaacgg agcatgagca ttttcgaagt tttttagatg cactagaaca aagcgtgttg 6060gcttcctctg agcccgcttt ccttatatac ccgcattctg cagccttaca gaatgttcta 6120gaaggtccta gatgcattcg tttgaaaata ctcccggtgg gtgcaaagag acgcagacgg 6180aaaatgtatc tgggtctctt tattgtgtac actacttttc catgtaccga atgtgagtcg 6240ccctcctttt gcaacaagca gctcgaatgt tctagaaaaa ggtggaaaat agtataaata 6300ccgttgaaaa taaataccga acaacatttg ctctaattgt gaaattagaa atcttcaaac 6360tataatcatc tcactggatc cccgggattg gccaaaggac ccaaaggtat gtttcgaatg 6420atactaacat aacatagaac attttcagga ggacccttgg 6460 3 8330 DNA ArtificialSequence Description of Artificial Sequenceplasmid DNA 3 gttgtcgtaaagagatgttt ttattttact ttacaccggg tcctctctct ctgccagcac 60 agctcagtgttggctgtgtg ctcgggctcc tgccaccggc ggcctcatct tcttcttctt 120 cttctctcctgctctcgctt atcacttctt cattcattct tattcctttt catcatcaaa 180 ctagcatttcttactttatt tatttttttc aattttcaat tttcagataa aaccaaacta 240 cttgggttacagccgtcaac agatccccgg gattggccaa aggacccaaa ggtatgtttc 300 gaatgatactaacataacat agaacatttt caggaggacc cttgcttgga gggtaccgga 360 tgactgctccaaagaagaag cgtaagctca tgaacacgat taacatcgct aagaacgact 420 tctctgacatcgaactggct gctatcccgt tcaacactct ggctgaccat tacggtgagc 480 gtttagctcgcgaacagttg gcccttgagc atgagtctta cgagatgggt gaagcacgct 540 tccgcaagatgtttgagcgt caacttaaag ctggtgaggt tgcggataac gctgccgcca 600 agcctctcatcactacccta ctccctaaga tgattgcacg catcaacgac tggtttgagg 660 aagtgaaagctaagcgcggc aagcgcccga cagccttcca gttcctgcaa gaaatcaagc 720 cggaagccgtagcgtacatc accattaaga ccactctggc ttgcctaacc agtgctgaca 780 atacaaccgttcaggctgta gcaagcgcaa tcggtcgggc cattgaggac gaggctcgct 840 tcggtcgtatccgtgacctt gaagctaagc acttcaagaa aaacgttgag gaacaactca 900 acaagcgcgtagggcacgtc tacaagaaag catttatgca agttgtcgag gctgacatgc 960 tctctaagggtctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa gactctattc 1020 atgtaggagtacgctgcatc gagatgctca ttgagtcaac cggaatggtt agcttacacc 1080 gccaaaatgctggcgtagta ggtcaagact ctgagactat cgaactcgca cctgaatacg 1140 ctgaggctatcgcaacccgt gcaggtgcgc tggctggcat ctctccgatg ttccaacctt 1200 gcgtagttcctcctaagccg tggactggca ttactggtgg tggctattgg gctaacggtc 1260 gtcgtcctctggcgctggtg cgtactcaca gtaagaaagc actgatgcgc tacgaagacg 1320 tttacatgcctgaggtgtac aaagcgatta acattgcgca aaacaccgca tggaaaatca 1380 acaagaaagtcctagcggtc gccaacgtaa tcaccaagtg gaagcattgt ccggtcgagg 1440 acatccctgcgattgagcgt gaagaactcc cgatgaaacc ggaagacatc gacatgaatc 1500 ctgaggctctcaccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag acaaggctcg 1560 caagtctcgccgtatcagcc ttgagttcat gcttgagcaa gccaataagt ttgctaacca 1620 taaggccatctggttccctt acaacatgga ctggcgcggt tcgtgtttac gctgtgtcaa 1680 tgttcaacccgcaaggtaac gatatgacca aaggacgtct tacgctggcg aaaggtaaac 1740 caatcggtaaggaaggttac tactggctga aaatccacgg tgcaaactgt gcgggtgtcg 1800 ataaggtttcgtttcctgag cgcatcaagt tcattgagga aaaccacgag aacatcatgg 1860 cttgcgctaagtctccactg gagaacactt ggtgggctga gcaagattct ccgttctgct 1920 tccttgcgttctgctttgag tacgctgggg tacagcacca cggcctgagc tataactgct 1980 cccttccgctggcgtttgac gggtcttgct ctggcatcca gcacttctcc gcgatgctcc 2040 gagatgaggtaggtggtcgc gcggttaact tgcttcctag tgaaaccgtt caggacatct 2100 acgggattgttgctaagaaa gtcaacgaga ttctgcaagc agacgcaatc aatgggaccg 2160 ataacgaagtagttaccgtg accgatgaga acactggtga aatctctgag aaagtcaagc 2220 tgggcactaaggcactggct ggtcaatggc tggcttacgg tgttactcgc agtgtgacta 2280 agcgttcagtcatgacgctg gcttacgggt ccaaagagtt cggcttccgt caacaagtgc 2340 tggaagataccattcagcca gctattgatt ccggcaaggg tctgatgttc actcagccga 2400 atcaggctgctggatacatg gctaagctga tttgggaatc cgtgagcgtg acggtggtag 2460 ctgcggttgaagcaatgaac tggcttaagt ctgctgctaa gctgctggct gctgaggtca 2520 aagataagaagactggagag attcttcgca agcgttgcgc tgtgcattgg gtaactcctg 2580 atggtttccctgtgtggcag gaatacaaga agcctattca gacgcgcttg aacctgatgt 2640 tcctcggtcagttccgctta cagcctacca ttaacaccaa caaagatagc gagattgatg 2700 cacacaaacaggagtctggt atcgctccta actttgtaca cagccaagac ggtagccacc 2760 ttcgtaagactgtagtgtgg gcacacgaga agtacggaat cgaatctttt gcactgattc 2820 acgactccttcggtaccatt ccggctgacg ctgcgaacct gttcaaagca gtgcgcgaaa 2880 ctatggttgacacatatgag tcttgtgatg tactggctga tttctacgac cagttcgctg 2940 accagttgcacgagtctcaa ttggacaaaa tgccagcact tccggctaaa ggtaacttga 3000 acctccgtgacatcttagag tcggacttcg cgttcgcgta agggcccact agtcggccgt 3060 acgggccctttcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3120 tcccggagacggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3180 gcgcgtcagcgggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3240 ttgtactgagagtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 3300 accgcatcaggcggccttaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 3360 ataataatggtttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 3420 atttgtttatttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 3480 taaatgcttcaataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3540 cttattcccttttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3600 aaagtaaaagatgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3660 aacagcggtaagatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3720 tttaaagttctgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3780 ggtcgccgcatacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3840 catcttacggatggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3900 aacactgcggccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3960 ttgcacaacatgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 4020 gccataccaaacgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 4080 aaactattaactggcgaact acttactcta gcttcccggc aacaattaat agactggatg 4140 gaggcggataaagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 4200 gctgataaatctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 4260 gatggtaagccctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 4320 gaacgaaatagacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 4380 gaccaagtttactcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 4440 atctaggtgaagatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4500 ttccactgagcgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4560 ctgcgcgtaatctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4620 ccggatcaagagctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4680 ccaaatactgtccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4740 ccgcctacatacctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4800 tcgtgtcttaccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4860 tgaacggggggttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4920 tacctacagcgtgagcattg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4980 tatccggtaagcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5040 gcctggtatctttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5100 tgatgctcgtcaggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5160 ttcctggccttttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5220 gtggataaccgtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5280 gagcgcagcgagtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5340 cccgcgcgttggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5400 ggcagtgagcgcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5460 cactttatgcttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5520 ggaaacagctatgaccatga ttacgccaag ctgtaagttt aaacatgatc ttactaacta 5580 actattctcatttaaatttt cagagcttaa aaatggctga aatcactcac aacgatggat 5640 acgctaacaacttggaaatg aaataagctt gcatgcctgc agagcaaaaa aatactgctt 5700 ttccttgcaaaattcggtgc tttcttcaaa gagaaacttt tgaagtcggc gcgagcattt 5760 ccttctttgacttctctctt tccgccaaaa agcctagcat ttttattgat aatttgatta 5820 cacacactcagagttcttcg acatgataaa gtgtttcatt ggcactcgcc ctaacagtac 5880 atgacaagggcggattatta tcgatcgata ttgaagacaa actccaaatg tgtgctcatt 5940 ttggagccccgtgtggggca gctgctctca atatattact agggagacga ggagggggac 6000 cttatcgaacgtcgcatgag ccattctttc ttctttatgc actctcttca ctctctcaca 6060 cattaatcgattcatagact cccatattcc ttgatgaagg tgtgggtttt tagctttttt 6120 tcccgatttgtaaaaggaag aggctgacga tgttaggaaa aagagaacgg agccgaaaaa 6180 acatccgtagtaagtcttcc ttttaagccg acacttttta gacagcattc gccgctagtt 6240 ttgaagtttaaattttaaaa aataaaaatt agtttcaatt ttttttaatt actaaatagg 6300 caaaagttttttcaagaact ctagaaaaac tagcttaatt catgggtact agaaaaattc 6360 ttgttttaaatttaatattt atcttaagat gtaattacga gaagcttttt tgaaaattct 6420 caattaaaagaatttgccga tttagaataa aagtcttcag aaatgagtaa aagctcaaat 6480 tagaagtttgtttttaaagg aaaaacacga aaaaagaaca ctatttatct tttcctcccc 6540 gcgtaaaattagttgttgtg ataatagtga tccgctgtct atttgcactc ggctcttcac 6600 accgtgcttcctctcacttg acccaacagg aaaaaaaaac atcacgtctg agacggtgaa 6660 ttgccttatcaagagcgtcg tctctttcac ccagtaacaa aaaaaatttg gtttctttac 6720 tttatatttatgtaggtcac aaaaaaaaag tgatgcagtt ttgtgggtcg gttgtctcca 6780 caccacctccgcctccagca gcacacaatc atcttcgtgt gttctcgacg attccttgta 6840 tgccgcggtcgtgaatgcac cacattcgac gcgcaactac acaccacact cactttcggt 6900 ggtattactacacgtcatcg ttgttcgtag tctcccgctc tttcgtcccc actcactcct 6960 cattattccccttggtgtat tgattttttt taaatggtac accactcctg acgtttctac 7020 cttcttgttttccgtccatt tagattttat ctggaaattt ttttaaaatt ttaggccaga 7080 gagttctagttcttgttcta aaagtctagg tcagacatac attttctatt tctcatcaaa 7140 aaaaaagttgataaagaaaa ctggttattc agaaagagtg tgtctcgttg aaattgattc 7200 aaaaaaaaattcccacccct cgcttgtttc tcaaaatatg agatcaacgg attttttcct 7260 tctcgattcaattttttgct gcgctctgtc tgccaaagtg tgtgtgtccg agcaaaagat 7320 gagagaatttacaaacagaa atgaaaaaaa gttggccaaa taatgaagtt ttatccgaga 7380 ttgatgggaaagatattaat gttctttacg gtttggaggg gagagagaga tagattttcg 7440 catcaaactccgccttttac atgtctttta gaatctaaaa tagatttttc tcatcatttt 7500 taatagaaaatcgagaaatt acagtaattt cgcaattttc ttgccaaaaa tacacgaaat 7560 ttgtgggtctcgccacgatc tcggtcttag tggttcattt ggtttaaaag tttataaaat 7620 ttcaaattctagtgtttaat ttccgcataa ttggacctaa aatgggtttt tgtcatcatt 7680 ttcaacaagaaatcgtgaaa atcctgttgt ttcgcaattt tcttttcaaa aatacacgaa 7740 atatatggtaatttcccgaa atattgaggg tctcgccacg atttcagtca cagtggccag 7800 gatttatcacgaaaaaagtt cgcctagtct cacatttccg gaaaaccgaa tctaaattag 7860 ttttttgtcatcattttgaa caaaaaatcg agacatccct atagtttcgc aattttcgtc 7920 gcttttctctccaaaaatga cagtctagaa ttaaaattcg ctggaactgg gaccatgata 7980 tcttttctccccgtttttca ttttattttt tattacactg gattgactaa aggtcaccac 8040 caccgccagtgtgtgccata tcacacacac acacacacac aatgtcgaga ttttatgtgt 8100 tatccctgcttgatttcgtt ccgttgtctc tctctctcta ttcatctttt gagccgagaa 8160 gctccagagaatggagcaca caggatcccg gcgcgcgatg tcgtcgggag atggcgccgc 8220 ctgggaagccgccgagagat atcagggaag atcgtctgat ttctcctcgg atgccacctc 8280 atctctcgagtttctccgcc tgttactccc tgccgaacct gatatttccc 8330 4 6470 DNA ArtificialSequence Description of Artificial Sequenceplasmid DNA 4 aagcttgcatgcctgcaggc cttggtcgac tctagacact tttcagctac ctagatacat 60 ggatatccccgcctcccaat ccacccaccc agggaaaaag aagggctcgc cgaaaaatca 120 aagttatctccaggctcgcg catcccaccg agcggttgac ttctctccac cacttttcat 180 tttaaccctcggggtacggg attggccaaa ggacccaaag gtatgtttcg aatgatacta 240 acataacatagaacattttc aggaggaccc ttgcttggag ggtaccgagc tcagaaaaaa 300 tgactgctccaaagaagaag cgtaaggtac cggtaatgaa cacgattaac atcgctaaga 360 acgacttctctgacatcgaa ctggctgcta tcccgttcaa cactctggct gaccattacg 420 gtgagcgtttagctcgcgaa cagttggccc ttgagcatga gtcttacgag atgggtgaag 480 cacgcttccgcaagatgttt gagcgtcaac ttaaagctgg tgaggttgcg gataacgctg 540 ccgccaagcctctcatcact accctactcc ctaagatgat tgcacgcatc aacgactggt 600 ttgaggaagtgaaagctaag cgcggcaagc gcccgacagc cttccagttc ctgcaagaaa 660 tcaagccggaagccgtagcg tacatcacca ttaagaccac tctggcttgc ctaaccagtg 720 ctgacaatacaaccgttcag gctgtagcaa gcgcaatcgg tcgggccatt gaggacgagg 780 ctcgcttcggtcgtatccgt gaccttgaag ctaagcactt caagaaaaac gttgaggaac 840 aactcaacaagcgcgtaggg cacgtctaca agaaagcatt tatgcaagtt gtcgaggctg 900 acatgctctctaagggtcta ctcggtggcg aggcgtggtc ttcgtggcat aaggaagact 960 ctattcatgtaggagtacgc tgcatcgaga tgctcattga gtcaaccgga atggttagct 1020 tacaccgccaaaatgctggc gtagtaggtc aagactctga gactatcgaa ctcgcacctg 1080 aatacgctgaggctatcgca acccgtgcag gtgcgctggc tggcatctct ccgatgttcc 1140 aaccttgcgtagttcctcct aagccgtgga ctggcattac tggtggtggc tattgggcta 1200 acggtcgtcgtcctctggcg ctggtgcgta ctcacagtaa gaaagcactg atgcgctacg 1260 aagacgtttacatgcctgag gtgtacaaag cgattaacat tgcgcaaaac accgcatgga 1320 aaatcaacaagaaagtccta gcggtcgcca acgtaatcac caagtggaag cattgtccgg 1380 tcgaggacatccctgcgatt gagcgtgaag aactcccgat gaaaccggaa gacatcgaca 1440 tgaatcctgaggctctcacc gcgtggaaac gtgctgccgc tgctgtgtac cgcaaggaca 1500 gggctcgcaagtctcgccgt atcagccttg agttcatgct tgagcaagcc aataagtttg 1560 ctaaccataaggccatctgg ttcccttaca acatggactg gcgcggtcgt gtttacgccg 1620 tgtcaatgttcaacccgcaa ggtaacgata tgaccaaagg actgcttacg ctggcgaaag 1680 gtaaaccaatcggtaaggaa ggttactact ggctgaaaat ccacggtgca aactgtgcgg 1740 gtgtcgataaggttccgttc cctgagcgca tcaagttcat tgaggaaaac cacgagaaca 1800 tcatggcttgcgctaagtct ccactggaga acacttggtg ggctgagcaa gattctccgt 1860 tctgcttccttgcgttctgc tttgagtacg ctggggtaca gcaccacggc ctgagctata 1920 actgctcccttccgctggcg tttgacgggt cttgctctgg catccagcac ttctccgcga 1980 tgctccgagatgaggtaggt ggtcgcgcgg ttaacttgct tcctagtgag accgttcagg 2040 acatctacgggattgttgct aagaaagtca acgagattct acaagcagac gcaatcaatg 2100 ggaccgataacgaagtagtt accgtgaccg atgagaacac tggtgaaatc tctgagaaag 2160 tcaagctgggcactaaggca ctggctggtc aatggctggc tcacggtgtt actcgcagtg 2220 tgactaagcgttcagtcatg acgctggctt acgggtccaa agagttcggc ttccgtcaac 2280 aagtgctggaagataccatt cagccagcta ttgattccgg caagggtccg atgttcactc 2340 agccgaatcaggctgctgga tacatggcta agctgatttg ggaatctgtg agcgtgacgg 2400 tggtagctgcggttgaagca atgaactggc ttaagtctgc tgctaagctg ctggctgctg 2460 aggtcaaagataagaagact ggagagattc ttcgcaagcg ttgcgctgtg cattgggtaa 2520 ctcctgatggtttccctgtg tggcaggaat acaagaagcc tattcagacg cgcttgaacc 2580 tgatgttcctcggtcagttc cgcttacagc ctaccattaa caccaacaaa gatagcgaga 2640 ttgatgcacacaaacaggag tctggtatcg ctcctaactt tgtacacagc caagacggta 2700 gccaccttcgtaagactgta gtgtgggcac acgagaagta cggaatcgaa tcttttgcac 2760 tgattcacgactccttcggt accattccgg ctgacgctgc gaacctgttc aaagcagtgc 2820 gcgaaactatggttgacaca tatgagtctt gtgatgtact ggctgatttc tacgaccagt 2880 tcgctgaccagttgcacgag tctcaattgg acaaaatgcc agcacttccg gctaaaggta 2940 acttgaacctccgtgacatc ttagagtcgg acttcgcgtt cgcgtaagaa ttccaactga 3000 gcgccggtcgctaccattac caacttgtct ggtgtcaaaa ataatagggg ccgctgtcat 3060 cagagtaagtttaaactgag ttctactaac taacgagtaa tatttaaatt ttcagcatct 3120 cgcgcccgtgcctctgactt ctaagtccaa ttactcttca acatccctac atgctctttc 3180 tccctgtgctcccaccccct atttttgtta ttatcaaaaa aacttcttct taatttcttt 3240 gttttttagcttcttttaag tcacctctaa caatgaaatt gtgtagattc aaaaatagaa 3300 ttaattcgtaataaaaagtc gaaaaaaatt gtgctccctc cccccattaa taataattct 3360 atcccaaaatctacacaatg ttctgtgtac acttcttatg ttttttttac ttctgataaa 3420 ttttttttgaaacatcatag aaaaaaccgc acacaaaata ccttatcata tgttacgttt 3480 cagtttatgaccgcaatttt tatttcttcg cacgtctggg cctctcatga cgtcaaatca 3540 tgctcatcgtgaaaaagttt tggagtattt ttggaatttt tcaatcaagt gaaagtttat 3600 gaaattaattttcctgcttt tgctttttgg gggtttcccc tattgtttgt caagagtttc 3660 gaggacggcgtttttcttgc taaaatcaca agtattgatg agcacgatgc aagaaagatc 3720 ggaagaaggtttgggtttga ggctcagtgg aaggtgagta gaagttgata atttgaaagt 3780 ggagtagtgtctatggggtt tttgccttaa atgacagaat acattcccaa tataccaaac 3840 ataactgtttcctactagtc ggccgtacgg gccctttcgt ctcgcgcgtt tcggtgatga 3900 cggtgaaaacctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 3960 tgccgggagcagacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg 4020 gcttaactatgcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 4080 accgcacagatgcgtaagga gaaaataccg catcaggcgg ccttaagggc ctcgtgatac 4140 gcctatttttataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 4200 ttcggggaaatgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 4260 atccgctcatgagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 4320 tgagtattcaacatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 4380 tttttgctcacccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 4440 gagtgggttacatcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 4500 aagaacgttttccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 4560 gtattgacgccgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 4620 ttgagtactcaccagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 4680 gcagtgctgccataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 4740 gaggaccgaaggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 4800 atcgttgggaaccggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 4860 ctgtagcaatggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4920 cccggcaacaattaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4980 cggcccttccggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 5040 gcggtatcattgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 5100 cgacggggagtcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 5160 cactgattaagcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 5220 taaaacttcatttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 5280 ccaaaatcccttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 5340 aaggatcttcttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 5400 caccgctaccagcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 5460 taactggcttcagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 5520 gccaccacttcaagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 5580 cagtggctgctgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 5640 taccggataaggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5700 agcgaacgacctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc 5760 ttcccgaagggagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5820 gcacgagggagcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5880 acctctgacttgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5940 acgccagcaacgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 6000 tctttcctgcgttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 6060 ataccgctcgccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 6120 agcgcccaatacgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 6180 acgacaggtttcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 6240 tcactcattaggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 6300 ttgtgagcggataacaattt cacacaggaa acagctatga ccatgattac gccaagctgt 6360 aagtttaaacatgatcttac taactaacta ttctcattta aattttcaga gcttaaaaat 6420 ggctgaaatcactcacaacg atggatacgc taacaacttg gaaatgaaat 6470 5 4689 DNA ArtificialSequence Description of Artificial Sequenceplasmid DNA 5 aagcttgcatgcctgcaggc cttggtcgac tctagacact tttcagctac ctagatacat 60 ggatatccccgcctcccaat ccacccaccc agggaaaaag aagggctcgc cgaaaaatca 120 aagttatctccaggctcgcg catcccaccg agcggttgac ttctctccac cacttttcat 180 tttaaccctcggggtacggg attggccaaa ggacccaaag gtatgtttcg aatgatacta 240 acataacatagaacattttc aggaggaccc ttgcttggag ggtaccgagc tcccgggatt 300 aatacgactcactataccgg tagaaaaaat gagtaaagga gaagaacttt tcactggagt 360 tgtcccaattcttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg 420 agagggtgaaggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg 480 aaaactacctgttccatggg taagtttaaa catatatata ctaactaacc ctgattattt 540 aaattttcagccaacacttg tcactacttt ctgttatggt gttcaatgct tctcgagata 600 cccagatcatatgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca 660 ggaaagaactatatttttca aagatgacgg gaactacaag acacgtaagt ttaaacagtt 720 cggtactaactaaccataca tatttaaatt ttcaggtgct gaagtcaagt ttgaaggtga 780 tacccttgttaatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct 840 tggacacaaattggaataca actataactc acacaatgta tacatcatgg cagacaaaca 900 aaagaatggaatcaaagttg taagtttaaa catgatttta ctaactaact aatctgattt 960 aaattttcagaacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga 1020 ccattatcaacaaaatactc caattggcga tggccctgtc cttttaccag acaaccatta 1080 cctgtccacacaatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct 1140 tcttgagtttgtaacagctg ctgggattac acatggcatg gatgaactat acaaatagca 1200 ttcgtagaattccaactgag cgccggtcgc taccattacc aacttgtctg gtgtcaaaaa 1260 taataggggccgctgtcatc agagtaagtt taaactgagt tctactaact aacgagtaat 1320 atttaaattttcagcatctc gcgcccgtgc ctctgacttc taagtccaat tactcttcaa 1380 catccctacatgctctttct ccctgtgctc ccacccccta tttttgttat tatcaaaaaa 1440 acttcttcttaatttctttg ttttttagct tcttttaagt cacctctaac aatgaaattg 1500 tgtagattcaaaaatagaat taattcgtaa taaaaagtcg aaaaaaattg tgctccctcc 1560 ccccattaataataattcta tcccaaaatc tacacaatgt tctgtgtaca cttcttatgt 1620 tttttttacttctgataaat tttttttgaa acatcataga aaaaaccgca cacaaaatac 1680 cttatcatatgttacgtttc agtttatgac cgcaattttt atttcttcgc acgtctgggc 1740 ctctcatgacgtcaaatcat gctcatcgtg aaaaagtttt ggagtatttt tggaattttt 1800 caatcaagtgaaagtttatg aaattaattt tcctgctttt gctttttggg ggtttcccct 1860 attgtttgtcaagagtttcg aggacggcgt ttttcttgct aaaatcacaa gtattgatga 1920 gcacgatgcaagaaagatcg gaagaaggtt tgggtttgag gctcagtgga aggtgagtag 1980 aagttgataatttgaaagtg gagtagtgtc tatggggttt ttgccttaaa tgacagaata 2040 cattcccaatataccaaaca taactgtttc ctactagtcg gccgtacggg ccctttcgtc 2100 tcgcgcgtttcggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 2160 cagcttgtctgtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 2220 ttggcgggtgtcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 2280 accatatgcggtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcggc 2340 cttaagggcctcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 2400 tagacgtcaggtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 2460 taaatacattcaaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 2520 tattgaaaaaggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 2580 gcggcattttgccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 2640 gaagatcagttgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2700 cttgagagttttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2760 tgtggcgcggtattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 2820 tattctcagaatgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 2880 atgacagtaagagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 2940 ttacttctgacaacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 3000 gatcatgtaactcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 3060 gagcgtgacaccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 3120 gaactacttactctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 3180 gcaggaccacttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 3240 gccggtgagcgtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3300 cgtatcgtagttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3360 atcgctgagataggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 3420 tatatactttagattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 3480 ctttttgataatctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 3540 gaccccgtagaaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 3600 tgcttgcaaacaaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 3660 ccaactctttttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 3720 ctagtgtagccgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 3780 gctctgctaatcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 3840 ttggactcaagacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 3900 tgcacacagcccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 3960 cattgagaaagcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4020 agggtcggaacaggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4080 agtcctgtcgggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4140 gggcggagcctatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4200 tggccttttgctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4260 accgcctttgagtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 4320 gtgagcgaggaagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4380 attcattaatgcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 4440 gcaattaatgtgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 4500 gctcgtatgttgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 4560 catgattacgccaagctgta agtttaaaca tgatcttact aactaactat tctcatttaa 4620 attttcagagcttaaaaatg gctgaaatca ctcacaacga tggatacgct aacaacttgg 4680 aaatgaaat4689 6 5175 DNA Artificial Sequence Description of ArtificialSequenceplasmid DNA 6 gatcccggcg cgcgatgtcg tcgggagatg gcgccgcctgggaagccgcc gagagatatc 60 agggaagatc gtctgatttc tcctcggatg ccacctcatctctcgagttt ctccgcctgt 120 tactccctgc cgaacctgat atttcccgtt gtcgtaaagagatgttttta ttttacttta 180 caccgggtcc tctctctctg ccagcacagc tcagtgttggctgtgtgctc gggctcctgc 240 caccggcggc ctcatcttct tcttcttctt ctctcctgctctcgcttatc acttcttcat 300 tcattcttat tccttttcat catcaaacta gcatttcttactttatttat ttttttcaat 360 tttcaatttt cagataaaac caaactactt gggttacagccgtcaacaga tccccgggat 420 tggccaaagg acccaaaggt atgtttcgaa tgatactaacataacataga acattttcag 480 gaggaccctt gcttggaggg taccggtaga aaaaatgagtaaaggagaag aacttttcac 540 tggagttgtc ccaattcttg ttgaattaga tggtgatgttaatgggcaca aattttctgt 600 cagtggagag ggtgaaggtg atgcaacata cggaaaacttacccttaaat ttatttgcac 660 tactggaaaa ctacctgttc catgggtaag tttaaacatatatatactaa ctaaccctga 720 ttatttaaat tttcagccaa cacttgtcac tactttctgttatggtgttc aatgcttctc 780 gagataccca gatcatatga aacggcatga ctttttcaagagtgccatgc ccgaaggtta 840 tgtacaggaa agaactatat ttttcaaaga tgacgggaactacaagacac gtaagtttaa 900 acagttcggt actaactaac catacatatt taaattttcaggtgctgaag tcaagtttga 960 aggtgatacc cttgttaata gaatcgagtt aaaaggtattgattttaaag aagatggaaa 1020 cattcttgga cacaaattgg aatacaacta taactcacacaatgtataca tcatggcaga 1080 caaacaaaag aatggaatca aagttgtaag tttaaacttggacttactaa ctaacggatt 1140 atatttaaat tttcagaact tcaaaattag acacaacattgaagatggaa gcgttcaact 1200 agcagaccat tatcaacaaa atactccaat tggcgatggccctgtccttt taccagacaa 1260 ccattacctg tccacacaat ctgccctttc gaaagatcccaacgaaaaga gagaccacat 1320 ggtccttctt gagtttgtaa cagctgctgg gattacacatggcatggatg aactatacaa 1380 atagcattcg tagaattcca actgagcgcc ggtcgctaccattaccaact tgtctggtgt 1440 caaaaataat aggggccgct gtcatcagag taagtttaaactgagttcta ctaactaacg 1500 agtaatattt aaattttcag catctcgcgc ccgtgcctctgacttctaag tccaattact 1560 cttcaacatc cctacatgct ctttctccct gtgctcccaccccctatttt tgttattatc 1620 aaaaaaactt cttcttaatt tctttgtttt ttagcttcttttaagtcacc tctaacaatg 1680 aaattgtgta gattcaaaaa tagaattaat tcgtaataaaaagtcgaaaa aaattgtgct 1740 ccctcccccc attaataata attctatccc aaaatctacacaatgttctg tgtacacttc 1800 ttatgttttt tttacttctg ataaattttt tttgaaacatcatagaaaaa accgcacaca 1860 aaatacctta tcatatgtta cgtttcagtt tatgaccgcaatttttattt cttcgcacgt 1920 ctgggcctct catgacgtca aatcatgctc atcgtgaaaaagttttggag tatttttgga 1980 atttttcaat caagtgaaag tttatgaaat taattttcctgcttttgctt tttgggggtt 2040 tcccctattg tttgtcaaga gtttcgagga cggcgtttttcttgctaaaa tcacaagtat 2100 tgatgagcac gatgcaagaa agatcggaag aaggtttgggtttgaggctc agtggaaggt 2160 gagtagaagt tgataatttg aaagtggagt agtgtctatggggtttttgc cttaaatgac 2220 agaatacatt cccaatatac caaacataac tgtttcctactagtcggccg tacgggcccg 2280 gtacccagct tttgttccct ttagtgaggg ttaattgcgcgcttggcgta atcatggtca 2340 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattccacacaacat acgagccgga 2400 agcataaagt gtaaagcctg gggtgcctaa tgagtgagctaactcacatt aattgcgttg 2460 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgccagctgcatta atgaatcggc 2520 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctcttccgcttcctc gctcactgac 2580 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcagctcactcaaa ggcggtaata 2640 cggttatcca cagaatcagg ggataacgca ggaaagaacatgtgagcaaa aggccagcaa 2700 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttttccataggct ccgcccccct 2760 gacgagcatc acaaaaatcg acgctcaagt cagaggtggcgaaacccgac aggactataa 2820 agataccagg cgtttccccc tggaagctcc ctcgtgcgctctcctgttcc gaccctgccg 2880 cttaccggat acctgtccgc ctttctccct tcgggaagcgtggcgctttc tcatagctca 2940 cgctgtaggt atctcagttc ggtgtaggtc gttcgctccaagctgggctg tgtgcacgaa 3000 ccccccgttc agcccgaccg ctgcgcctta tccggtaactatcgtcttga gtccaacccg 3060 gtaagacacg acttatcgcc actggcagca gccactggtaacaggattag cagagcgagg 3120 tatgtaggcg gtgctacaga gttcttgaag tggtggcctaactacggcta cactagaagg 3180 acagtatttg gtatctgcgc tctgctgaag ccagttaccttcggaaaaag agttggtagc 3240 tcttgatccg gcaaacaaac caccgctggt agcggtggtttttttgtttg caagcagcag 3300 attacgcgca gaaaaaaagg atctcaagaa gatcctttgatcttttctac ggggtctgac 3360 gctcagtgga acgaaaactc acgttaaggg attttggtcatgagattatc aaaaaggatc 3420 ttcacctaga tccttttaaa ttaaaaatga agttttaaatcaatctaaag tatatatgag 3480 taaacttggt ctgacagtta ccaatgctta atcagtgaggcacctatctc agcgatctgt 3540 ctatttcgtt catccatagt tgcctgactc cccgtcgtgtagataactac gatacgggag 3600 ggcttaccat ctggccccag tgctgcaatg ataccgcgagacccacgctc accggctcca 3660 gatttatcag caataaacca gccagccgga agggccgagcgcagaagtgg tcctgcaact 3720 ttatccgcct ccatccagtc tattaattgt tgccgggaagctagagtaag tagttcgcca 3780 gttaatagtt tgcgcaacgt tgttgccatt gctacaggcatcgtggtgtc acgctcgtcg 3840 tttggtatgg cttcattcag ctccggttcc caacgatcaaggcgagttac atgatccccc 3900 atgttgtgca aaaaagcggt tagctccttc ggtcctccgatcgttgtcag aagtaagttg 3960 gccgcagtgt tatcactcat ggttatggca gcactgcataattctcttac tgtcatgcca 4020 tccgtaagat gcttttctgt gactggtgag tactcaaccaagtcattctg agaatagtgt 4080 atgcggcgac cgagttgctc ttgcccggcg tcaatacgggataataccgc gccacatagc 4140 agaactttaa aagtgctcat cattggaaaa cgttcttcggggcgaaaact ctcaaggatc 4200 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtgcacccaactg atcttcagca 4260 tcttttactt tcaccagcgt ttctgggtga gcaaaaacaggaaggcaaaa tgccgcaaaa 4320 aagggaataa gggcgacacg gaaatgttga atactcatactcttcctttt tcaatattat 4380 tgaagcattt atcagggtta ttgtctcatg agcggatacatatttgaatg tatttagaaa 4440 aataaacaaa taggggttcc gcgcacattt ccccgaaaagtgccacctaa attgtaagcg 4500 ttaatatttt gttaaaattc gcgttaaatt tttgttaaatcagctcattt tttaaccaat 4560 aggccgaaat cggcaaaatc ccttataaat caaaagaatagaccgagata gggttgagtg 4620 ttgttccagt ttggaacaag agtccactat taaagaacgtggactccaac gtcaaagggc 4680 gaaaaaccgt ctatcagggc gatggcccac tacgtgaaccatcaccctaa tcaagttttt 4740 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaaagggagcccc cgatttagag 4800 cttgacgggg aaagccggcg aacgtggcga gaaaggaagggaagaaagcg aaaggagcgg 4860 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgtaaccaccaca cccgccgcgc 4920 ttaatgcgcc gctacagggc gcgtcccatt cgccattcaggctgcgcaac tgttgggaag 4980 ggcgatcggt gcgggcctct tcgctattac gccagctggcgaaaggggga tgtgctgcaa 5040 ggcgattaag ttgggtaacg ccagggtttt cccagtcacgacgttgtaaa acgacggcca 5100 gtgagcgcgc gtaatacgac tcactatagg gcgaattggagctccaccgc ggtggcggcc 5160 gctctagaac tagtg 5175 7 12482 DNA ArtificialSequence Description of Artificial Sequenceplasmid DNA 7 gatcctccaaaatcgtcttc cgctctgaaa aacgaaagtg gacctttgac atccgaaaaa 60 atgggcgaaaaaatgaaatt gagctttttg ggtcgaaaaa aatgttttta gaatgctgag 120 aacacgttaaacacgaagat catatttatt ttgagacccg gatgctctga aaatgtctga 180 catagatttaaaaaagcata tatatatttt tcattttcaa cgtgaaagtt ttgtgcaact 240 ttatagaatctcctattggc acattgtttt ttatttaact gaggcagttt ttgaacacct 300 ttttgaaactttgaatctct ttgaagtata ctgtcgaaaa gactgacttg agcgttcgaa 360 atgccagaagaaaactatat ttgaatctcg cgctaaattg agaaatgcaa ccgcgctcca 420 ctggacaattggaaaaaaaa tttattcgga ggcgacaacg gtattttcga aattgatttt 480 ctgtgtattttctcattttt tataaattct tctttgattt atcgttcgtt tgtgagaaat 540 ttaattgtattcaaactttt ttatagtaag ataccggtgg taccgctagc cgtacgaacc 600 cgggattggccaaaggaccc aaaggtatgt ttcgaatgat actaacataa catagaacat 660 tttcaggaggacccttgctt ggagggtacc ggatgactgc tccaaagaag aagcgtaagc 720 tcatgaacacgattaacatc gctaagaacg acttctctga catcgaactg gctgctatcc 780 cgttcaacactctggctgac cattacggtg agcgtttagc tcgcgaacag ttggcccttg 840 agcatgagtcttacgagatg ggtgaagcac gcttccgcaa gatgtttgag cgtcaactta 900 aagctggtgaggttgcggat aacgctgccg ccaagcctct catcactacc ctactcccta 960 agatgattgcacgcatcaac gactggtttg aggaagtgaa agctaagcgc ggcaagcgcc 1020 cgacagccttccagttcctg caagaaatca agccggaagc cgtagcgtac atcaccatta 1080 agaccactctggcttgccta accagtgctg acaatacaac cgttcaggct gtagcaagcg 1140 caatcggtcgggccattgag gacgaggctc gcttcggtcg tatccgtgac cttgaagcta 1200 agcacttcaagaaaaacgtt gaggaacaac tcaacaagcg cgtagggcac gtctacaaga 1260 aagcatttatgcaagttgtc gaggctgaca tgctctctaa gggtctactc ggtggcgagg 1320 cgtggtcttcgtggcataag gaagactcta ttcatgtagg agtacgctgc atcgagatgc 1380 tcattgagtcaaccggaatg gttagcttac accgccaaaa tgctggcgta gtaggtcaag 1440 actctgagactatcgaactc gcacctgaat acgctgaggc tatcgcaacc cgtgcaggtg 1500 cgctggctggcatctctccg atgttccaac cttgcgtagt tcctcctaag ccgtggactg 1560 gcattactggtggtggctat tgggctaacg gtcgtcgtcc tctggcgctg gtgcgtactc 1620 acagtaagaaagcactgatg cgctacgaag acgtttacat gcctgaggtg tacaaagcga 1680 ttaacattgcgcaaaacacc gcatggaaaa tcaacaagaa agtcctagcg gtcgccaacg 1740 taatcaccaagtggaagcat tgtccggtcg aggacatccc tgcgattgag cgtgaagaac 1800 tcccgatgaaaccggaagac atcgacatga atcctgaggc tctcaccgcg tggaaacgtg 1860 ctgccgctgctgtgtaccgc aagacaaggc tcgcaagtct cgccgtatca gccttgagtt 1920 catgcttgagcaagccaata agtttgctaa ccataaggcc atctggttcc cttacaacat 1980 ggactggcgcggttcgtgtt tacgctgtgt caatgttcaa cccgcaaggt aacgatatga 2040 ccaaaggacgtcttacgctg gcgaaaggta aaccaatcgg taaggaaggt tactactggc 2100 tgaaaatccacggtgcaaac tgtgcgggtg tcgataaggt ttcgtttcct gagcgcatca 2160 agttcattgaggaaaaccac gagaacatca tggcttgcgc taagtctcca ctggagaaca 2220 cttggtgggctgagcaagat tctccgttct gcttccttgc gttctgcttt gagtacgctg 2280 gggtacagcaccacggcctg agctataact gctcccttcc gctggcgttt gacgggtctt 2340 gctctggcatccagcacttc tccgcgatgc tccgagatga ggtaggtggt cgcgcggtta 2400 acttgcttcctagtgaaacc gttcaggaca tctacgggat tgttgctaag aaagtcaacg 2460 agattctgcaagcagacgca atcaatggga ccgataacga agtagttacc gtgaccgatg 2520 agaacactggtgaaatctct gagaaagtca agctgggcac taaggcactg gctggtcaat 2580 ggctggcttacggtgttact cgcagtgtga ctaagcgttc agtcatgacg ctggcttacg 2640 ggtccaaagagttcggcttc cgtcaacaag tgctggaaga taccattcag ccagctattg 2700 attccggcaagggtctgatg ttcactcagc cgaatcaggc tgctggatac atggctaagc 2760 tgatttgggaatccgtgagc gtgacggtgg tagctgcggt tgaagcaatg aactggctta 2820 agtctgctgctaagctgctg gctgctgagg tcaaagataa gaagactgga gagattcttc 2880 gcaagcgttgcgctgtgcat tgggtaactc ctgatggttt ccctgtgtgg caggaataca 2940 agaagcctattcagacgcgc ttgaacctga tgttcctcgg tcagttccgc ttacagccta 3000 ccattaacaccaacaaagat agcgagattg atgcacacaa acaggagtct ggtatcgctc 3060 ctaactttgtacacagccaa gacggtagcc accttcgtaa gactgtagtg tgggcacacg 3120 agaagtacggaatcgaatct tttgcactga ttcacgactc cttcggtacc attccggctg 3180 acgctgcgaacctgttcaaa gcagtgcgcg aaactatggt tgacacatat gagtcttgtg 3240 atgtactggctgatttctac gaccagttcg ctgaccagtt gcacgagtct caattggaca 3300 aaatgccagcacttccggct aaaggtaact tgaacctccg tgacatctta gagtcggact 3360 tcgcgttcgcgtaagggccc tcgtcgagtc ggtcacaatc acctgaaact ccaaaggcag 3420 ccagtgaggaacgtgaagaa gaagaaaaag agtcatctga acaggtttga ttttctttct 3480 ggtcaaaaagatgaaattat tgattttcag ccagatactc ccaaaactag cagcgagaag 3540 tctgcaagtcgttcacagtc gcccagagaa tcgcgggaag tgagccaaga ggtatgtttt 3600 tcaaaaatcaataactgatc ataattttta ttgtttggtg aatttaagaa aataatattc 3660 gaaaattcctctgaattatc aagattgcag tattaatttc gagaaaaatt gagatattca 3720 tagagctattgtaaattttc ttgatttcag actgaaactt cggaaaatca agagaaaatc 3780 aaagaaaaggatgacgggga tgatcagcct ggcacaccga acagctatag aagccgggaa 3840 acttcaccagctccaaaaag gtccaaggag accaggtttg tcaaaagctt cctgcgatta 3900 attctcatttcaatttttca gagaatcaga gtctcctgaa aaatccccgg ttcgttcaag 3960 atctcccagaaggtcttcag cacgttcccc gtcacgatct cctagacggc gccgagaaag 4020 aagctcagaaagaaagcaat ccgaagagcc agcaccgcta ccagagaaaa agaagaaaga 4080 gccgctggatattctacgaa caagaaccgg aggagcatat attccacccg ccaaacttcg 4140 acttatgcaacaacagatta gtgataagca aagtgaacag tatcagagaa tgaattggga 4200 aagaatgaagaaaaagattc acggattggt taacagagtc aacgcgaaga atcttgttca 4260 aattgtcagagaacttcttc aagagaatgt gattcgttca aagtgagtga gaaaatcgaa 4320 ggaaaaggaaagaattaatt taatttttca ggggacttct ctgccgtgac attattcaag 4380 ctcaggctttctcaccagga ttctctaacg tctatgcagc tttggcggca gttatcaact 4440 cgaaattccctcatgtcggt gaacttcttc tccgtcgtct gattgtacag ttcaaaagaa 4500 gtttccgtagaaatgacaga ggcgtcacgg tgaacgtgat caaattcatc gcacatttga 4560 ttaatcaacaagttgctcac gaagttcttg cgctggaaat catgattctg atgcttgaag 4620 aaccaactgatgattcagtt gaagtcgcca ttgcgttcct gaaagagtgt ggagcaaagc 4680 ttctggagattgctccagca gctcttaaca gtgtctacga ccgtcttcgt gcaattctca 4740 tggaaactgaaagatcggaa aatgcactgg atcgacgtat tcagtatatg attgagactg 4800 caatgcagattcgaaaggac aaatttgcgg taaggtagaa tatataaata gtttattaga 4860 aaaaaataaattagaataat ttaaattcct actagccaat caggcgacct ttttgcgcat 4920 agttctattattgaaaaatt tggagaattt ctcatattct cgctcggaaa tctggaattc 4980 gacgagatcttctggcttct gtgcagctgc atcgctttgt gctccctttc tcgcttgtct 5040 tctgtgtacaccaagaacct tgttgagttc atcaactgaa tctgtgactg gcttgttgct 5100 cactggatgcactagacgac tgattctcga gaaatcagat tgagttgcga ttagggtgac 5160 ctagaaattgggaataatac gaacttttga aaatattcag gaggattaaa aaaattattc 5220 tcgacaatcctacaaattta cttattgcac catgttgctc caacattttt cattaaaagt 5280 taatgaaaaaatgtagaaaa tcggaaattg gcaattttca gaccattttt aagcattttc 5340 aaaaaaaaattgcagctgaa ataaatgtca ttttcagata aatcgagcga ttttctgttg 5400 tctgacactagtttttagtt ttaaaaaatg ttggaagaac atggtgcaat aggtaatttc 5460 atagaatttccatgtgtttt ttttcaatta accaattatc caaatcttcc aaactcacat 5520 tttgcggagctgggctatca agaatctgct gcagttttat aagacgagca tctctgatat 5580 cactgaaaattaatttttaa tcaaaacttg aatatcaact aaacccactt attaactttc 5640 tcgatcttctgtcgttcggt acgatgacgg tgaagaagcc aattgtagta gttgatttgg 5700 ttcaagtcctttcggtgttg tacgtcagtg tcctgcaatg ctatttagtt ataacttagg 5760 cctaagattcaatttaatga agtgattaaa tttgttctct gaacctctta agatgatctt 5820 ttggattagaaacatataag acaggtttac ctatctatta aaaaacagat caaaatagat 5880 acgaccaaatcggataatcc atgcctacct ggcatctagg aacgtgttct tagaagattt 5940 cttacgtaatcgtatgaaga aataacaatt tgatcgttgg ccagcaaaaa tagggtttta 6000 agtgggatagtgtttttatt agctaaccgg aaaattttat agtttttttt tgcaagaaac 6060 cactgaaaaccccctaattg tatacatttt ttggagcagc ttctggtctt tttgagcaat 6120 aaaattcgataaaacagaat ttaagtgtaa attgttcaca tttagtttct attttatcaa 6180 attttgttgctcaaaaacat tcgaagctgc tctaaaaaaa tgcattaaaa aaggggtttt 6240 cagtggtttttcacattaaa aaagctaatt ttaactaaaa atccatcata tttccaactt 6300 tgtcacaacaataaaatgct ggtcaaaatg tgttcgaaaa aatgtttttt tttttaattt 6360 ttataatttaaaaatagttt tctttcgctg ggacacatac atttttgggc gtaaattttc 6420 agttcaaatttccattttta caaccataat cataaagcta cgtctgatct ctctcgcact 6480 tacctgcgcctgattcgaaa gaacaaccgt agccaaaaga acaagaagaa caagcacgta 6540 gttgtggtagtggacgttca tcacgcaata ctgaccaatg gtcgtggggt ctcactttcc 6600 gtactattgagagaggggag actgaagatg gcaattgagg acagtgtctt cgacgcacgc 6660 atgcatccataagcataatc caggagggat ggagagaaaa atcttgtttc taagcccctc 6720 cctttgtaatacatacacat atctaatacc gaagaatggc taattgaatg gacgtcagct 6780 gttgctgtagttgccaaggc atcatcgatg aaataactga aagaaagaat taaataatta 6840 ttgcaggcgtatccggcggt cattgaagac ttggacttga ttgaggagga ggatcagatc 6900 atccatacacttaatttgga ggatgcggtt gatccggaaa atgggcttag taagtgactg 6960 accacacgcggggggcatta atttaataaa ttgaattcca tttcagatgt gttcaaacta 7020 gatccagaattcgaaaagaa cgaggaggtt tatgaggaga tccgtaagga aatcattgga 7080 aacgccgatatttcggatga ggatggtggc gacgagttgg atgatgaaga agagggtagt 7140 gatgtggaagaggctccgaa gaagactaca gagattattg ataatactga tcagaattga 7200 ctgctttcagaaggtattca ttttgagttt tgggccggca aatctgtaag ttgccggttg 7260 ccgaaaatttgctgaatttg ccggaaaaaa aaattccgga atttatttaa aaactttttg 7320 taaaaattaaattaaatttg caacttttca gagaagtcta cctgacaatg caatcatctt 7380 tggactaccaagaagctgct cacaaattgc tgaaaatgaa gattccagac agcatgcagg 7440 tcagcgatgttgcaaagaaa aattttcgac caaaaaaacc aaccaatcat aaaatttaaa 7500 aaaaaactccgtttttttct ttttttttat acgagaaaaa ccaaaaaaat gtatttttgc 7560 caaattctaaaatactatcc ccgaaatttt caatattttc tctttcagaa cgaactctgc 7620 gcgatgcttgtcgattgttg tgctcaacag cgtacctacg agcgattcta cggaatgctc 7680 atcgaacgtttctgccgact tcgcctcgaa taccagcaat actttgaaaa gctctgccag 7740 gacacgtattccacgattca ccgaattgac atcacaaaac tgcggaattt ggctcgcctt 7800 attgctcatttgctctcgac ggatgctatt gactggaaga ttttggccga tatgaaaatg 7860 accgaagaggacacaacttc ttctggcaga atctatatta aatatatatt taatgaactt 7920 gtggaggcgatgggaatggt taaacttcat tcgagagtta ctgatccgtg agtttcctag 7980 agagagttgttttcgtattc aattttccct attttcagaa ctttggctca ttgctttgtt 8040 ggattattcccacgaactaa tccgaacagc gcacgatttt cgatcaactt cttcacaatg 8100 attggattgggtggtttgac gttggaactt cgtgaatggc tggcaaaggg tctcaagaag 8160 aagaagggaatgctggatca gttgaaggcc gaatcaagct cagattcatc gtcgtcttcg 8220 gattcgtcagactcgtctga ttcttcggat tctgacgatt catccgactc gtcttcagat 8280 tcctcatcttcttcagaatc agagccagaa ccaccgaaga aaaagaagaa gaagaacagt 8340 gaagagagttccaaaaagaa ggaaaaagag aatattggtc gacgggatcg tggagacaag 8400 agagctgaacgtcatcgtga tcaaagtgtg gagaacaagg acaaggatcg tcgacgtcgc 8460 caggattctgacgaaaatcg tcggccagaa cgaggagatg accgcaagga tcggagtaaa 8520 gatcgtcgtcgtcaagactc ggatgatgag gatcggaaag gtcgtgaacg tcgggaagat 8580 tcaggggaaagacgtcgcgg agatcgggat cgacgtgatc gaaacaagga tcaggaggat 8640 caccgtgaagatcgccgtga ccgaagcaag gatcgtgagg atcgacgtga tcgccgtcgt 8700 catgactctgatgatgatcg taaaactcgt cgggatagaa gtgaagagcg aggaggacgt 8760 cgtcgtgaagtggaatcgga tgatcgacgc cgacgtcgtt gaattttcaa attttaaata 8820 ctgaatatttgttttttttc ctattattta tttattctct ttgtgttttt tttcttgctt 8880 tctaaaaaattaattcaatc caaatctaaa catgagcggt tttttttctc tttccgtctc 8940 ccaattcgtattccgctcct ctcatctgaa cacaatgtgc aagtttattt atcttctcgc 9000 tttcatttcattaggacgtg gggggaattg gtggaagggg gaaacacaca aaaggatgat 9060 ggaaatgaaataaggacaca caatatgcaa caacattcaa ttcagaaata tggaggaagg 9120 tttaaaagaaaacataaaaa tatatagagg aggaaggaaa actagtaaaa aataagcaaa 9180 gaaattaggcgaacgatgag aattgtcctc gcttggcaaa tgcgaatccg tatggagagg 9240 cacgtttggcgaaggcaaat gttcggtatg gagatctgta aaaattttta agttgaaatt 9300 tggtgttgctcttttacaaa attttccgat tttcgcttga aattacggtg ccaggtctcg 9360 acacgtcttccaatttttca aattcaaaag agcctttaat gggctgtagt tgctaatttc 9420 tcgtttttgaaaatttttct tccgtttaat cgaaatttga tgtattttat ttatgatttt 9480 caataaatttcaaagaaact ggtgaaaact cggaaaattg tgaactacag taatccaatc 9540 cttaaaggcgcacacctttt aaatgtccgc cccaatacga tattttttta agattcgcta 9600 gagcggccgccaccgcggtg gagctccaat tcgccctata gtgagtcgta ttacaattca 9660 ctggccgtcgttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 9720 cttgcagcacatcccccctt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 9780 ccttcccaacagttgcgtag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 9840 agcgcggcgggtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 9900 cccgctcctttcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 9960 gctctaaatcgggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 10020 aaaaaacttgattagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 10080 cgccctttgacgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 10140 acactcaaccctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 10200 tattggttaaaaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 10260 acgtttacaatttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 10320 tttttctaaatacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 10380 caataatattgaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 10440 ttttttgcggcattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 10500 gatgctgaagatcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 10560 aagatccttgagagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 10620 ctgctatgtggcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 10680 atacactattctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 10740 gatggcatgacagtaagaga attatgcagt gctgccataa gcatgagtga taacactgcg 10800 gccaacttacttctgacaac gatcggagga ccgaaggagc taaccgcttt ttttcacaac 10860 atgggggatcatgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 10920 aacgacgagcgtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 10980 actggcgaactacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 11040 aaagttgcaggaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 11100 tctggagccggtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 11160 ccctcccgtatcgtagttat ctacacgacg ggcagtcagg caactatgga tgaacgaaat 11220 agacagatcgctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 11280 tactcatatatactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 11340 aagatcctttttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 11400 gcgtcagaccccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 11460 atctgctgcttgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 11520 gagctaccaactctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 11580 gtccttctagtgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 11640 tacctcgctctgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 11700 accgggttggactcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 11760 ggttcgtgcacacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 11820 cgtgagcattgagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 11880 agcggcagggtcggaacagg agagcgcacg agggagcttc caggggggaa cgcctggtat 11940 ctttatagtcctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 12000 tcaggggggccgagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 12060 ttttgctggccttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 12120 cgtattaccgcctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 12180 gagtcagtgagcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 12240 tggccgattcattaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 12300 cgcaacgcaattaatgtgag ttacctcact cattaggcac cccaggcttt acactttatg 12360 cttccggctcctatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 12420 tatgaccatgattacgccaa gctcggaatt aaccctcact aaagggaaca aaagctgggg 12480 gg 12482 87209 DNA Artificial Sequence Description of Artificial SequenceplasmidDNA 8 gatccgtcga cagatctccc tatagtgagt cgtattactg cagccaagct aattccgggc60 gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata 120caaattttaa agtgactctt aggttttaaa acgaaaattc ttgttcttga gtaactcttt 180cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc gctcttattg accacacctc 240taccggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 300ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 360taatgagtga ggtaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 420aacctgtcgt gccagctgga ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 480attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 540cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 600gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 660ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 720agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 780tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 840ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 900gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 960ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1020gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 1080aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 1140aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 1200ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 1260gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 1320gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 1380tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 1440ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 1500ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 1560atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 1620ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 1680tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 1740attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 1800tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 1860ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 1920gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 1980gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 2040gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 2100aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 2160taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 2220tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 2280tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2340atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2400tttccccgaa aagtgccacc tgaacgaagc atctgtgctt cattttgtag aacaaaaatg 2460caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa 2520atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa 2580aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa 2640cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc 2700tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt 2760ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa 2820ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac 2880ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc 2940cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg 3000ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata 3060ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct 3120tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc 3180gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc 3240acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat 3300attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag 3360cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg 3420gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc 3480gcacatacag ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat 3540acatgagaag aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt 3600tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta 3660tcgtatgctt ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg 3720attagtctca tccttcaatg ctatcatttc ctttgatatt ggatcatatt aagaaaccat 3780tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg 3840tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 3900tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 3960gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag 4020atcaacgaca ttactatata tataatatag gaagcattta atagacagca tcgtaatata 4080tgtgtacttt gcagttatga cgccagatgg cagtagtgga agatattctt tattgaaaaa 4140tagcttgtca ccttacgtac aatcttgatc cggagctttt ctttttttgc cgattaagaa 4200ttaattcggt cgaaaaaaga aaaggagagg gccaagaggg agggcattgg tgactattga 4260gcacgtgagt atacgtgatt aagcacacaa aggcagcttg gagtatgtct gttattaatt 4320tcacaggtag ttctggtcca ttggtgaaag tttgcggctt gcagagcaca gaggccgcag 4380aatgtgctct agattccgat gctgacttgc tgggtattat atgtgtgccc aatagaaaga 4440gaacaattga cccggttatt gcaaggaaaa tttcaagtct tgtaaaagca tataaaaata 4500gttcaggcac tccgaaatac ttggttggcg tgtttcgtaa tcaacctaag gaggatgttt 4560tggctctggt caatgattac ggcattgata tcgtccaact gcatggagat gagtcgtggc 4620aagaatacca agagttcctc ggtttgccag ttattaaaag actcgtattt ccaaaagact 4680gcaacatact actcagtgca gcttcacaga aacctcattc gtttattccc ttgtttgatt 4740cagaagcagg tgggacaggt gaacttttgg attggaactc gatttctgac tgggttggaa 4800ggcaagagag ccccgaaagc ttacatttta tgttagctgg tggactgacg ccagaaaatg 4860ttggtgatgc gcttagatta aatggcgtta ttggtgttga tgtaagcgga ggtgtggaga 4920caaatggtgt aaaagactct aacaaaatag caaatttcgt caaaaatgct aagaaatagg 4980ttattactga gtagtattta tttaagtatt gtttgtgcac ttgccgatct atgcggtgtg 5040aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa acgttaatat 5100tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 5160aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 5220agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 5280cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 5340gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 5400gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 5460ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 5520gccgctacag ggcgcgtcgc gccattcgcc attcaggctg cgcaactgtt gggaagggcg 5580atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 5640attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtcg 5700tccaagcttt cgcgagctcg agatcccgag ctttgcaaat taaagccttc gagcgtccca 5760aaaccttctc aagcaaggtt ttcagtataa tgttacatgc gtacacgcgt ctgtacagaa 5820aaaaaagaaa aatttgaaat ataaataacg ttcttaatac taacataact ataaaaaaat 5880aaatagggac ctagacttca ggttgtctaa ctccttcctt ttcggttaga gcggatgtgg 5940ggggagggcg tgaatgtaag cgtgacataa ctaattacat gatatccttt tgttgtttcc 6000gggtgtacaa tatggacttc ctcttttctg gcaaccaaac ccatacatcg ggattcctat 6060aataccttcg ttggtctccc taacatgtag gtggcggagg ggagatatac aatagaacag 6120ataccagaca agacataatg ggctaaacaa gactacacca attacactgc ctcattgatg 6180gtggtacata acgaactaat actgtagccc tagacttgat agccatcatc atatcgaagt 6240ttcactaccc tttttccatt tgccatctat tgaagtaata ataggcgcat gcaacttctt 6300ttcttttttt ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa 6360aaaatgatgg aagacactaa aggaaaaaat taacgacaaa gacagcacca acagatgtcg 6420ttgttccaga gctgatgagg ggtatcttcg aacacacgaa actttttcct tccttcattc 6480acgcacacta ctctctaatg agcaacggta tacggccttc cttccagtta cttgaatttg 6540aaataaaaaa agtttgccgc tttgctatca agtataaata gacctgcaat tattaatctt 6600ttgtttcctc gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt 6660tcaagctata ccaagcatac aatcaactcc aagcttgaag caagcctcct gaaagatgaa 6720gctactgtct tctatcgaac aagcatgcga tatttgccga cttaaaaagc tcaagtgctc 6780caaagaaaaa ccgaagtgcg ccaagtgtct gaagaacaac tgggagtgtc gctactctcc 6840caaaaccaaa aggtctccgc tgactagggc acatctgaca gaagtggaat caaggctaga 6900aagactggaa cagctatttc tactgatttt tcctcgagaa gaccttgaca tgattttgaa 6960aatggattct ttacaggata taaaagcatt gttaacagga ttatttgtac aagataatgt 7020gaataaagat gccgtcacag atagattggc ttcagtggag actgatatgc ctctaacatt 7080gagacagcat agaataagtg cgacatcatc atcggaagag agtagtaaca aaggtcaaag 7140acagttgact gtatcgccgg aattcttaat acgactcact atagggcata tggccatgga 7200ggccccggg 7209 9 6820 DNA Artificial Sequence Description of ArtificialSequenceplasmid DNA 9 gatccgtcga cagatctccc tatagtgagt cgtattactgcagagatcta tgaatcgtag 60 atactgaaaa accccgcaag ttcacttcaa ctgtgcatcgtgcaccatct caatttcttt 120 catttataca tcgttttgcc ttcttttatg taactatactcctctaagtt tcaatcttgg 180 ccatgtaacc tctgatctat agaatttttt aaatgactagaattaatgcc catctttttt 240 ttggacctaa attcttcatg aaaatatatt acgagggcttattcagaagc tttggacttc 300 ttcgccagag gtttggtcaa gtctccaatc aaggttgtcggcttgtctac cttgccagaa 360 atttacgaaa agatggaaaa gggtcaaatc gttggtagatacgttgttga cacttctaaa 420 taagcgaatt tcttatgatt tatgattttt attattaaataagttataaa aaaaataagt 480 gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaaaattcttgtt cttgagtaac 540 tctttcctgt aggtcaggtt gctttctcag gtatagcatgaggtcgctct tattgaccac 600 acctctaccg gcatgcccga aattccccta ccctatgaacatattccatt ttgtaatttc 660 gtgtcgtttc tattatgaat ttcatttata aagtttatgtacaaatatca taaaaaaaga 720 gaatcttttt aagcaaggat tttcttaact tcttcggcgacagcatcacc gacttcggtg 780 gtactgttgg aaccacctaa atcaccagtt ctgatacctgcatccaaaac ctttttaact 840 gcatcttcaa tggccttacc ttcttcaggc aagttcaatgacaatttcaa catcattgca 900 gcagacaaga tagtggcgat agggtcaacc ttattctttggcaaatctgg agcagaaccg 960 tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctggcaaagaggc caaggacgca 1020 gatggcaaca aacccaagga acctgggata acggaggcttcatcggagat gatatcacca 1080 aacatgttgc tggtgattat aataccattt aggtgggttgggttcttaac taggatcatg 1140 gcggcagaat caatcaattg atgttgaacc ttcaatgtaggaaattcgtt cttgatggtt 1200 tcctccacag tttttctcca taatcttgaa gaggccaaaacattagcttt atccaaggac 1260 caaataggca atggtggctc atgttgtagg gccatgaaagcggccattct tgtgattctt 1320 tgcacttctg gaacggtgta ttgttcacta tcccaagcgacaccatcacc atcgtcttcc 1380 tttctcttac caaagtaaat acctcccact aattctctgacaacaacgaa gtcagtacct 1440 ttagcaaatt gtggcttgat tggagataag tctaaaagagagtcggatgc aaagttacat 1500 ggtcttaagt tggcgtacaa ttgaagttct ttacggatttttagtaaacc ttgttcaggt 1560 ctaacactac ctgtacccca tttaggacca cccacagcacctaacaaaac ggcatcaacc 1620 ttcttggagg cttccagcgc ctcatctgga agtgggacacctgtagcatc gatagcagca 1680 ccaccaatta aatgattttc gaaatcgaac ttgacattggaacgaacatc agaaatagct 1740 ttaagaacct taatggcttc ggctgtgatt tcttgaccaacgtggtcacc tggcaaaacg 1800 acgatcttct taggggcaga cattagaatg gtatatccttgaaatatata tatatattgc 1860 tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgattgctaaccac ctattggaaa 1920 aaacaatagg tccttaaata atattgtcaa cttcaagtattgtgatgcaa gcatttagtc 1980 atgaacgctt ctctattcta tatgaaaagc cggttccggcctctcacctt tcctttttct 2040 cccaattttt cagttgaaaa aggtatatgc gtcaggcgacctctgaaatt aacaaaaaat 2100 ttccagtcat cgaatttgat tctgtgcgat agcgcccctgtgtgttctcg ttatgttgag 2160 gaaaaaaata atggttgcta agagattcga actcttgcatcttacgatac ctgagtattc 2220 ccacagttgg ggatctcgac tctagctaga ggatcaattcgtaatcatgg tcatagctgt 2280 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaacatacgagcc ggaagcataa 2340 agtgtaaagc ctggggtgcc taatgagtga ggtaactcacattaattgcg ttgcgctcac 2400 tgcccgcttt ccagtcggga aacctgtcgt gccagctggattaatgaatc ggccaacgcg 2460 cggggagagg cggtttgcgt attgggcgct cttccgcttcctcgctcact gactcgctgc 2520 gctcggtcgt tcggctgcgg cgagcggtat cagctcactcaaaggcggta atacggttat 2580 ccacagaatc aggggataac gcaggaaaga acatgtgagcaaaaggccag caaaaggcca 2640 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccataggctccgcccc cctgacgagc 2700 atcacaaaaa tcgacgctca agtcagaggt ggcgaaacccgacaggacta taaagatacc 2760 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgttccgaccctg ccgcttaccg 2820 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgctttctcatagc tcacgctgta 2880 ggtatctcag ttcggtgtag gtcgttcgct ccaagctgggctgtgtgcac gaaccccccg 2940 ttcagcccga ccgctgcgcc ttatccggta actatcgtcttgagtccaac ccggtaagac 3000 acgacttatc gccactggca gcagccactg gtaacaggattagcagagcg aggtatgtag 3060 gcggtgctac agagttcttg aagtggtggc ctaactacggctacactaga aggacagtat 3120 ttggtatctg cgctctgctg aagccagtta ccttcggaaaaagagttggt agctcttgat 3180 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgtttgcaagcag cagattacgc 3240 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttctacggggtct gacgctcagt 3300 ggaacgaaaa ctcacgttaa gggattttgg tcatgagattatcaaaaagg atcttcacct 3360 agatcctttt aaattaaaaa tgaagtttta aatcaatctaaagtatatat gagtaaactt 3420 ggtctgacag ttaccaatgc ttaatcagtg aggcacctatctcagcgatc tgtctatttc 3480 gttcatccat agttgcctga ctccccgtcg tgtagataactacgatacgg gagggcttac 3540 catctggccc cagtgctgca atgataccgc gagacccacgctcaccggct ccagatttat 3600 cagcaataaa ccagccagcc ggaagggccg agcgcagaagtggtcctgca actttatccg 3660 cctccatcca gtctattaat tgttgccggg aagctagagtaagtagttcg ccagttaata 3720 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggtgtcacgctcg tcgtttggta 3780 tggcttcatt cagctccggt tcccaacgat caaggcgagttacatgatcc cccatgttgt 3840 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgtcagaagtaag ttggccgcag 3900 tgttatcact catggttatg gcagcactgc ataattctcttactgtcatg ccatccgtaa 3960 gatgcttttc tgtgactggt gagtactcaa ccaagtcattctgagaatag tgtatgcggc 4020 gaccgagttg ctcttgcccg gcgtcaatac gggataataccgcgccacat agcagaactt 4080 taaaagtgct catcattgga aaacgttctt cggggcgaaaactctcaagg atcttaccgc 4140 tgttgagatc cagttcgatg taacccactc gtgcacccaactgatcttca gcatctttta 4200 ctttcaccag cgtttctggg tgagcaaaaa caggaaggcaaaatgccgca aaaaagggaa 4260 taagggcgac acggaaatgt tgaatactca tactcttcctttttcaatat tattgaagca 4320 tttatcaggg ttattgtctc atgagcggat acatatttgaatgtatttag aaaaataaac 4380 aaataggggt tccgcgcaca tttccccgaa aagtgccacctgacgtctaa gaaaccatta 4440 ttatcatgac attaacctat aaaaataggc gtatcacgaggccctttcgt ctcgcgcgtt 4500 tcggtgatga cggtgaaaac ctctgacaca tgcagctcccggagacggtc acagcttgtc 4560 tgtaagcgga tgccgggagc agacaagccc gtcagggcgcgtcagcgggt gttggcgggt 4620 gtcggggctg gcttaactat gcggcatcag agcagattgtactgagagtg caccataacg 4680 catttaagca taaacacgca ctatgccgtt cttctcatgtatatatatat acaggcaaca 4740 cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgcgcagctcgcg ttgcattttc 4800 ggaagcgctc gttttcggaa acgctttgaa gttcctattccgaagttcct attctctagc 4860 tagaaagtat aggaacttca gagcgctttt gaaaaccaaaagcgctctga agacgcactt 4920 tcaaaaaacc aaaaacgcac cggactgtaa cgagctactaaaatattgcg aataccgctt 4980 ccacaaacat tgctcaaaag tatctctttg ctatatatctctgtgctata tccctatata 5040 acctacccat ccacctttcg ctccttgaac ttgcatctaaactcgacctc tacatttttt 5100 atgtttatct ctagtattac tctttagaca aaaaaattgtagtaagaact attcatagag 5160 tgaatcgaaa acaatacgaa aatgtaaaca tttcctatacgtagtatata gagacaaaat 5220 agaagaaacc gttcataatt ttctgaccaa tgaagaatcatcaacgctat cactttctgt 5280 tcacaaagta tgcgcaatcc acatcggtat agaatataatcggggatgcc tttatcttga 5340 aaaaatgcac ccgcagcttc gctagtaatc agtaaacgcgggaagtggag tcaggctttt 5400 tttatggaag agaaaataga caccaaagta gccttcttctaaccttaacg gacctacagt 5460 gcaaaaagtt atcaagagac tgcattatag agcgcacaaaggagaaaaaa agtaatctaa 5520 gatgctttgt tagaaaaata gcgctctcgg gatgcatttttgtagaacaa aaaagaagta 5580 tagattcttt gttggtaaaa tagcgctctc gcgttgcatttctgttctgt aaaaatgcag 5640 ctcagattct ttgtttgaaa aattagcgct ctcgcgttgcatttttgttt tacaaaaatg 5700 aagcacagat tcttcgttgg taaaatagcg ctttcgcgttgcatttctgt tctgtaaaaa 5760 tgcagctcag attctttgtt tgaaaaatta gcgctctcgcgttgcatttt tgttctacaa 5820 aatgaagcac agatgcttcg ttgcttgcat gcaacttcttttcttttttt ttcttttctc 5880 tctcccccgt tgttgtctca ccatatccgc aatgacaaaaaaaatgatgg aagacactaa 5940 aggaaaaaat taacgacaaa gacagcacca acagatgtcgttgttccaga gctgatgagg 6000 ggtatcttcg aacacacgaa actttttcct tccttcattcacgcacacta ctctctaatg 6060 agcaacggta tacggccttc cttccagtta cttgaatttgaaataaaaaa agtttgccgc 6120 tttgctatca agtataaata gacctgcaat tattaatcttttgtttcctc gtcattgttc 6180 tcgttccctt tcttccttgt ttctttttct gcacaatatttcaagctata ccaagcatac 6240 aatcaactcc aagctttgca aagatggata aagcggaattaattcccgag cctccaaaaa 6300 agaagagaaa ggtcgaattg ggtaccgccg ccaattttaatcaaagtggg aatattgctg 6360 atagctcatt gtccttcact ttcactaaca gtagcaacggtccgaacctc ataacaactc 6420 aaacaaattc tcaagcgctt tcacaaccaa ttgcctcctctaacgttcat gataacttca 6480 tgaataatga aatcacggct agtaaaattg atgatggtaataattcaaaa ccactgtcac 6540 ctggttggac ggaccaaact gcgtataacg cgtttggaatcactacaggg atgtttaata 6600 ccactacaat ggatgatgta tataactatc tattcgatgatgaagatacc ccaccaaacc 6660 caaaaaaaga gatcgaattc ttaatacgac tcactatagggcccatggac gaagaatcca 6720 gttcattctt atgtacctat gctgagaatc gtgccaataagaagccaata cttccttaga 6780 tgatgcaata aatattaaaa taaaacaaaa cagaaggctg6820 10 10597 DNA Artificial Sequence Description of ArtificialSequenceplasmid DNA 10 ccggtggtac cgggcccccc ctcgaggtcg acggtatcgataagctttcg tcattgaaaa 60 gaaggataag aatggacgat gggaagaagc tctcgttgttccaggagatc agaaaacagc 120 aactgttcca aatcttaagg agggagaaga atatcaattcagaatttctg ctcgtaacaa 180 ggctggaact ggagatcctt ctgatccttc tgatcgtgttgttgcgaagc caagaaacct 240 tgctccaaga attcatcgtg aagatctttc tgatacaactgtcaaggtcg gagccactct 300 caagttcatt gttcatattg atggtgagcc agcaccagatgtaacatggt cattcaatgg 360 aaaaggaatc ggagagagca aggctcaaat tgaaaatgagccatacatct cgagatttgc 420 tttgccaaag gcacttcgta agcaaagtgg aaaatataccatcactgcaa ccaacattaa 480 tggaactgac agtgtcacta tcaatatcaa ggtaaaaagcaagccaacga aaccaaaggg 540 accaatcgag gtaactgatg tcttcgaaga tcgtgcaactcttgactgga aaccaccaga 600 ggatgacgga ggagagccaa ttgagttcta tgaaattgaaaagatgaaca ccaaggacgg 660 aatctgggtt ccatgtggac gtagtggaga tacccacttcacagtcgatt cactcaacaa 720 gggagatcat tacaagttcc gtgtcaaggc tgtcaacagcgaaggacctt ctgatccatt 780 ggaaactgaa accgatattt tggctaaaaa tccatttgatcgtccagata gaccaggtcg 840 tccagagcca actgattggg attctgatca tgttgatctcaagtgggatc cactagttct 900 agaagcgctg ctaagggggc cctcgtcgag tcggtcacaatcacctgaaa ctccaaaggc 960 agccagtgag gaacgtgaag aagaagaaaa agagtcatctgaacaggttt gattttcttt 1020 ctggtcaaaa agatgaaatt attgattttc agccagatactcccaaaact agcagcgaga 1080 agtctgcaag tcgttcacag tcgcccagag aatcgcgggaagtgagccaa gaggtatgtt 1140 tttcaaaaat caataactga tcataatttt tattgtttggtgaatttaag aaaataatat 1200 tcgaaaattc ctctgaatta tcaagattgc agtattaatttcgagaaaaa ttgagatatt 1260 catagagcta ttgtaaattt tcttgatttc agactgaaacttcggaaaat caagagaaaa 1320 tcaaagaaaa ggatgacggg gatgatcagc ctggcacaccgaacagctat agaagccggg 1380 aaacttcacc agctccaaaa aggtccaagg agaccaggtttgtcaaaagc ttcctgcgat 1440 taattctcat ttcaattttt cagagaatca gagtctcctgaaaaatcccc ggttcgttca 1500 agatctccca gaaggtcttc agcacgttcc ccgtcacgatctcctagacg gcgccgagaa 1560 agaagctcag aaagaaagca atccgaagag ccagcaccgctaccagagaa aaagaagaaa 1620 gagccgctgg atattctacg aacaagaacc ggaggagcatatattccacc cgccaaactt 1680 cgacttatgc aacaacagat tagtgataag caaagtgaacagtatcagag aatgaattgg 1740 gaaagaatga agaaaaagat tcacggattg gttaacagagtcaacgcgaa gaatcttgtt 1800 caaattgtca gagaacttct tcaagagaat gtgattcgttcaaagtgagt gagaaaatcg 1860 aaggaaaagg aaagaattaa tttaattttt caggggacttctctgccgtg acattattca 1920 agctcaggct ttctcaccag gattctctaa cgtctatgcagctttggcgg cagttatcaa 1980 ctcgaaattc cctcatgtcg gtgaacttct tctccgtcgtctgattgtac agttcaaaag 2040 aagtttccgt agaaatgaca gaggcgtcac ggtgaacgtgatcaaattca tcgcacattt 2100 gattaatcaa caagttgctc acgaagttct tgcgctggaaatcatgattc tgatgcttga 2160 agaaccaact gatgattcag ttgaagtcgc cattgcgttcctgaaagagt gtggagcaaa 2220 gcttctggag attgctccag cagctcttaa cagtgtctacgaccgtcttc gtgcaattct 2280 catggaaact gaaagatcgg aaaatgcact ggatcgacgtattcagtata tgattgagac 2340 tgcaatgcag attcgaaagg acaaatttgc ggtaaggtagaatatataaa tagtttatta 2400 gaaaaaaata aattagaata atttaaattc ctactagccaatcaggcgac ctttttgcgc 2460 atagttctat tattgaaaaa tttggagaat ttctcatattctcgctcgga aatctggaat 2520 tcgacgagat cttctggctt ctgtgcagct gcatcgctttgtgctccctt tctcgcttgt 2580 cttctgtgta caccaagaac cttgttgagt tcatcaactgaatctgtgac tggcttgttg 2640 ctcactggat gcactagacg actgattctc gagaaatcagattgagttgc gattagggtg 2700 acctagaaat tgggaataat acgaactttt gaaaatattcaggaggatta aaaaaattat 2760 tctcgacaat cctacaaatt tacttattgc accatgttgctccaacattt ttcattaaaa 2820 gttaatgaaa aaatgtagaa aatcggaaat tggcaattttcagaccattt ttaagcattt 2880 tcaaaaaaaa attgcagctg aaataaatgt cattttcagataaatcgagc gattttctgt 2940 tgtctgacac tagtttttag ttttaaaaaa tgttggaagaacatggtgca ataggtaatt 3000 tcatagaatt tccatgtgtt ttttttcaat taaccaattatccaaatctt ccaaactcac 3060 attttgcgga gctgggctat caagaatctg ctgcagttttataagacgag catctctgat 3120 atcactgaaa attaattttt aatcaaaact tgaatatcaactaaacccac ttattaactt 3180 tctcgatctt ctgtcgttcg gtacgatgac ggtgaagaagccaattgtag tagttgattt 3240 ggttcaagtc ctttcggtgt tgtacgtcag tgtcctgcaatgctatttag ttataactta 3300 ggcctaagat tcaatttaat gaagtgatta aatttgttctctgaacctct taagatgatc 3360 ttttggatta gaaacatata agacaggttt acctatctattaaaaaacag atcaaaatag 3420 atacgaccaa atcggataat ccatgcctac ctggcatctaggaacgtgtt cttagaagat 3480 ttcttacgta atcgtatgaa gaaataacaa tttgatcgttggccagcaaa aatagggttt 3540 taagtgggat agtgttttta ttagctaacc ggaaaattttatagtttttt tttgcaagaa 3600 accactgaaa accccctaat tgtatacatt ttttggagcagcttctggtc tttttgagca 3660 ataaaattcg ataaaacaga atttaagtgt aaattgttcacatttagttt ctattttatc 3720 aaattttgtt gctcaaaaac attcgaagct gctctaaaaaaatgcattaa aaaaggggtt 3780 ttcagtggtt tttcacatta aaaaagctaa ttttaactaaaaatccatca tatttccaac 3840 tttgtcacaa caataaaatg ctggtcaaaa tgtgttcgaaaaaatgtttt tttttttaat 3900 ttttataatt taaaaatagt tttctttcgc tgggacacatacatttttgg gcgtaaattt 3960 tcagttcaaa tttccatttt tacaaccata atcataaagctacgtctgat ctctctcgca 4020 cttacctgcg cctgattcga aagaacaacc gtagccaaaagaacaagaag aacaagcacg 4080 tagttgtggt agtggacgtt catcacgcaa tactgaccaatggtcgtggg gtctcacttt 4140 ccgtactatt gagagagggg agactgaaga tggcaattgaggacagtgtc ttcgacgcac 4200 gcatgcatcc ataagcataa tccaggaggg atggagagaaaaatcttgtt tctaagcccc 4260 tccctttgta atacatacac atatctaata ccgaagaatggctaattgaa tggacgtcag 4320 ctgttgctgt agttgccaag gcatcatcga tgaaataactgaaagaaaga attaaataat 4380 tattgcaggc gtatccggcg gtcattgaag acttggacttgattgaggag gaggatcaga 4440 tcatccatac acttaatttg gaggatgcgg ttgatccggaaaatgggctt agtaagtgac 4500 tgaccacacg cggggggcat taatttaata aattgaattccatttcagat gtgttcaaac 4560 tagatccaga attcgaaaag aacgaggagg tttatgaggagatccgtaag gaaatcattg 4620 gaaacgccga tatttcggat gaggatggtg gcgacgagttggatgatgaa gaagagggta 4680 gtgatgtgga agaggctccg aagaagacta cagagattattgataatact gatcagaatt 4740 gactgctttc agaaggtatt cattttgagt tttgggccggcaaatctgta agttgccggt 4800 tgccgaaaat ttgctgaatt tgccggaaaa aaaaattccggaatttattt aaaaactttt 4860 tgtaaaaatt aaattaaatt tgcaactttt cagagaagtctacctgacaa tgcaatcatc 4920 tttggactac caagaagctg ctcacaaatt gctgaaaatgaagattccag acagcatgca 4980 ggtcagcgat gttgcaaaga aaaattttcg accaaaaaaaccaaccaatc ataaaattta 5040 aaaaaaaact ccgttttttt cttttttttt atacgagaaaaaccaaaaaa atgtattttt 5100 gccaaattct aaaatactat ccccgaaatt ttcaatattttctctttcag aacgaactct 5160 gcgcgatgct tgtcgattgt tgtgctcaac agcgtacctacgagcgattc tacggaatgc 5220 tcatcgaacg tttctgccga cttcgcctcg aataccagcaatactttgaa aagctctgcc 5280 aggacacgta ttccacgatt caccgaattg acatcacaaaactgcggaat ttggctcgcc 5340 ttattgctca tttgctctcg acggatgcta ttgactggaagattttggcc gatatgaaaa 5400 tgaccgaaga ggacacaact tcttctggca gaatctatattaaatatata tttaatgaac 5460 ttgtggaggc gatgggaatg gttaaacttc attcgagagttactgatccg tgagtttcct 5520 agagagagtt gttttcgtat tcaattttcc ctattttcagaactttggct cattgctttg 5580 ttggattatt cccacgaact aatccgaaca gcgcacgattttcgatcaac ttcttcacaa 5640 tgattggatt gggtggtttg acgttggaac ttcgtgaatggctggcaaag ggtctcaaga 5700 agaagaaggg aatgctggat cagttgaagg ccgaatcaagctcagattca tcgtcgtctt 5760 cggattcgtc agactcgtct gattcttcgg attctgacgattcatccgac tcgtcttcag 5820 attcctcatc ttcttcagaa tcagagccag aaccaccgaagaaaaagaag aagaagaaca 5880 gtgaagagag ttccaaaaag aaggaaaaag agaatattggtcgacgggat cgtggagaca 5940 agagagctga acgtcatcgt gatcaaagtg tggagaacaaggacaaggat cgtcgacgtc 6000 gccaggattc tgacgaaaat cgtcggccag aacgaggagatgaccgcaag gatcggagta 6060 aagatcgtcg tcgtcaagac tcggatgatg aggatcggaaaggtcgtgaa cgtcgggaag 6120 attcagggga aagacgtcgc ggagatcggg atcgacgtgatcgaaacaag gatcaggagg 6180 atcaccgtga agatcgccgt gaccgaagca aggatcgtgaggatcgacgt gatcgccgtc 6240 gtcatgactc tgatgatgat cgtaaaactc gtcgggatagaagtgaagag cgaggaggac 6300 gtcgtcgtga agtggaatcg gatgatcgac gccgacgtcgttgaattttc aaattttaaa 6360 tactgaatat ttgttttttt tcctattatt tatttattctctttgtgttt tttttcttgc 6420 tttctaaaaa attaattcaa tccaaatcta aacatgagcggttttttttc tctttccgtc 6480 tcccaattcg tattccgctc ctctcatctg aacacaatgtgcaagtttat ttatcttctc 6540 gctttcattt cattaggacg tggggggaat tggtggaagggggaaacaca caaaaggatg 6600 atggaaatga aataaggaca cacaatatgc aacaacattcaattcagaaa tatggaggaa 6660 ggtttaaaag aaaacataaa aatatataga ggaggaaggaaaactagtaa aaaataagca 6720 aagaaattag gcgaacgatg agaattgtcc tcgcttggcaaatgcgaatc cgtatggaga 6780 ggcacgtttg gcgaaggcaa atgttcggta tggagatctgtaaaaatttt taagttgaaa 6840 tttggtgttg ctcttttaca aaattttccg attttcgcttgaaattacgg tgccaggtct 6900 cgacacgtct tccaattttt caaattcaaa agagcctttaatgggctgta gttgctaatt 6960 tctcgttttt gaaaattttt cttccgttta atcgaaatttgatgtatttt atttatgatt 7020 ttcaataaat ttcaaagaaa ctggtgaaaa ctcggaaaattgtgaactac agtaatccaa 7080 tccttaaagg cgcacacctt ttaaatgtcc gccccaatacgatatttttt taagattcgc 7140 tagagcggcc gccaccgcgg tggagctcca attcgccctatagtgagtcg tattacaatt 7200 cactggccgt cgttttacaa cgtcgtgact gggaaaaccctggcgttacc caacttaatc 7260 gccttgcagc acatcccccc ttcgccagct ggcgtaatagcgaagaggcc cgcaccgatc 7320 gcccttccca acagttgcgt agcctgaatg gcgaatgggacgcgccctgt agcggcgcat 7380 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgctacacttgcc agcgccctag 7440 cgcccgctcc tttcgctttc ttcccttcct ttctcgccacgttcgccggc tttccccgtc 7500 aagctctaaa tcgggggctc cctttagggt tccgatttagtgctttacgg cacctcgacc 7560 ccaaaaaact tgattagggt gatggttcac gtagtgggccatcgccctga tagacggttt 7620 ttcgcccttt gacgttggag tccacgttct ttaatagtggactcttgttc caaactggaa 7680 caacactcaa ccctatctcg gtctattctt ttgatttataagggattttg ccgatttcgg 7740 cctattggtt aaaaaatgag ctgatttaac aaaaatttaacgcgaatttt aacaaaatat 7800 taacgtttac aatttcaggt ggcacttttc ggggaaatgtgcgcggaacc cctatttgtt 7860 tatttttcta aatacattca aatatgtatc cgctcatgagacaataaccc tgataaatgc 7920 ttcaataata ttgaaaaagg aagagtatga gtattcaacatttccgtgtc gcccttattc 7980 ccttttttgc ggcattttgc cttcctgttt ttgctcacccagaaacgctg gtgaaagtaa 8040 aagatgctga agatcagttg ggtgcacgag tgggttacatcgaactggat ctcaacagcg 8100 gtaagatcct tgagagtttt cgccccgaag aacgttttccaatgatgagc acttttaaag 8160 ttctgctatg tggcgcggta ttatcccgta ttgacgccgggcaagagcaa ctcggtcgcc 8220 gcatacacta ttctcagaat gacttggttg agtactcaccagtcacagaa aagcatctta 8280 cggatggcat gacagtaaga gaattatgca gtgctgccataagcatgagt gataacactg 8340 cggccaactt acttctgaca acgatcggag gaccgaaggagctaaccgct ttttttcaca 8400 acatggggga tcatgtaact cgccttgatc gttgggaaccggagctgaat gaagccatac 8460 caaacgacga gcgtgacacc acgatgcctg tagcaatggcaacaacgttg cgcaaactat 8520 taactggcga actacttact ctagcttccc ggcaacaattaatagactgg atggaggcgg 8580 ataaagttgc aggaccactt ctgcgctcgg cccttccggctggctggttt attgctgata 8640 aatctggagc cggtgagcgt gggtctcgcg gtatcattgcagcactgggg ccagatggta 8700 agccctcccg tatcgtagtt atctacacga cgggcagtcaggcaactatg gatgaacgaa 8760 atagacagat cgctgagata ggtgcctcac tgattaagcattggtaactg tcagaccaag 8820 tttactcata tatactttag attgatttaa aacttcatttttaatttaaa aggatctagg 8880 tgaagatcct ttttgataat ctcatgacca aaatcccttaacgtgagttt tcgttccact 8940 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttgagatcctttt tttctgcgcg 9000 taatctgctg cttgcaaaca aaaaaaccac cgctaccagcggtggtttgt ttgccggatc 9060 aagagctacc aactcttttt ccgaaggtaa ctggcttcagcagagcgcag ataccaaata 9120 ctgtccttct agtgtagccg tagttaggcc accacttcaagaactctgta gcaccgccta 9180 catacctcgc tctgctaatc ctgttaccag tggctgctgccagtggcgat aagtcgtgtc 9240 ttaccgggtt ggactcaaga cgatagttac cggataaggcgcagcggtcg ggctgaacgg 9300 ggggttcgtg cacacagccc agcttggagc gaacgacctacaccgaactg agatacctac 9360 agcgtgagca ttgagaaagc gccacgcttc ccgaagggagaaaggcggac aggtatccgg 9420 taagcggcag ggtcggaaca ggagagcgca cgagggagcttccagggggg aacgcctggt 9480 atctttatag tcctgtcggg tttcgccacc tctgacttgagcgtcgattt ttgtgatgct 9540 cgtcaggggg gccgagccta tggaaaaacg ccagcaacgcggccttttta cggttcctgg 9600 ccttttgctg gccttttgct cacatgttct ttcctgcgttatcccctgat tctgtggata 9660 accgtattac cgcctttgag tgagctgata ccgctcgccgcagccgaacg accgagcgca 9720 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacgcaaaccgcct ctccccgcgc 9780 gttggccgat tcattaatgc agctggcacg acaggtttcccgactggaaa gcgggcagtg 9840 agcgcaacgc aattaatgtg agttacctca ctcattaggcaccccaggct ttacacttta 9900 tgcttccggc tcctatgttg tgtggaattg tgagcggataacaatttcac acaggaaaca 9960 gctatgacca tgattacgcc aagctcggaa ttaaccctcactaaagggaa caaaagctgg 10020 gggggatcct ccaaaatcgt cttccgctct gaaaaacgaaagtggacctt tgacatccga 10080 aaaaatgggc gaaaaaatga aattgagctt tttgggtcgaaaaaaatgtt tttagaatgc 10140 tgagaacacg ttaaacacga agatcatatt tattttgagacccggatgct ctgaaaatgt 10200 ctgacataga tttaaaaaag catatatata tttttcattttcaacgtgaa agttttgtgc 10260 aactttatag aatctcctat tggcacattg ttttttatttaactgaggca gtttttgaac 10320 acctttttga aactttgaat ctctttgaag tatactgtcgaaaagactga cttgagcgtt 10380 cgaaatgcca gaagaaaact atatttgaat ctcgcgctaaattgagaaat gcaaccgcgc 10440 tccactggac aattggaaaa aaaatttatt cggaggcgacaacggtattt tcgaaattga 10500 ttttctgtgt attttctcat tttttataaa ttcttctttgatttatcgtt cgtttgtgag 10560 aaatttaatt gtattcaaac ttttttatag taagata10597 11 10599 DNA Artificial Sequence Description of ArtificialSequenceplasmid DNA 11 ccggtggtac cgctagccgt acgaacccgg gttctagaactagtggatcc cacttgagat 60 caacatgatc agaatcccaa tcagttggct ctggacgacctggtctatct ggacgatcaa 120 atggattttt agccaaaata tcggtttcag tttccaatggatcagaaggt ccttcgctgt 180 tgacagcctt gacacggaac ttgtaatgat ctcccttgttgagtgaatcg actgtgaagt 240 gggtatctcc actacgtcca catggaaccc agattccgtccttggtgttc atcttttcaa 300 tttcatagaa ctcaattggc tctcctccgt catcctctggtggtttccag tcaagagttg 360 cacgatcttc gaagacatca gttacctcga ttggtccctttggtttcgtt ggcttgcttt 420 ttaccttgat attgatagtg acactgtcag ttccattaatgttggttgca gtgatggtat 480 attttccact ttgcttacga agtgcctttg gcaaagcaaatctcgagatg tatggctcat 540 tttcaatttg agccttgctc tctccgattc cttttccattgaatgaccat gttacatctg 600 gtgctggctc accatcaata tgaacaatga acttgagagtggctccgacc ttgacagttg 660 tatcagaaag atcttcacga tgaattcttg gagcaaggtttcttggcttc gcaacaacac 720 gatcagaagg atcagaagga tctccagttc cagccttgttacgagcagaa attctgaatt 780 gatattcttc tccctcctta agatttggaa cagttgctgttttctgatct cctggaacaa 840 cgagagcttc ttcccatcgt ccattcttat ccttcttttcaatgacgaaa gcttatcgat 900 accgtcgacc tcgagggggg gccctcgtcg agtcggtcacaatcacctga aactccaaag 960 gcagccagtg aggaacgtga agaagaagaa aaagagtcatctgaacaggt ttgattttct 1020 ttctggtcaa aaagatgaaa ttattgattt tcagccagatactcccaaaa ctagcagcga 1080 gaagtctgca agtcgttcac agtcgcccag agaatcgcgggaagtgagcc aagaggtatg 1140 tttttcaaaa atcaataact gatcataatt tttattgtttggtgaattta agaaaataat 1200 attcgaaaat tcctctgaat tatcaagatt gcagtattaatttcgagaaa aattgagata 1260 ttcatagagc tattgtaaat tttcttgatt tcagactgaaacttcggaaa atcaagagaa 1320 aatcaaagaa aaggatgacg gggatgatca gcctggcacaccgaacagct atagaagccg 1380 ggaaacttca ccagctccaa aaaggtccaa ggagaccaggtttgtcaaaa gcttcctgcg 1440 attaattctc atttcaattt ttcagagaat cagagtctcctgaaaaatcc ccggttcgtt 1500 caagatctcc cagaaggtct tcagcacgtt ccccgtcacgatctcctaga cggcgccgag 1560 aaagaagctc agaaagaaag caatccgaag agccagcaccgctaccagag aaaaagaaga 1620 aagagccgct ggatattcta cgaacaagaa ccggaggagcatatattcca cccgccaaac 1680 ttcgacttat gcaacaacag attagtgata agcaaagtgaacagtatcag agaatgaatt 1740 gggaaagaat gaagaaaaag attcacggat tggttaacagagtcaacgcg aagaatcttg 1800 ttcaaattgt cagagaactt cttcaagaga atgtgattcgttcaaagtga gtgagaaaat 1860 cgaaggaaaa ggaaagaatt aatttaattt ttcaggggacttctctgccg tgacattatt 1920 caagctcagg ctttctcacc aggattctct aacgtctatgcagctttggc ggcagttatc 1980 aactcgaaat tccctcatgt cggtgaactt cttctccgtcgtctgattgt acagttcaaa 2040 agaagtttcc gtagaaatga cagaggcgtc acggtgaacgtgatcaaatt catcgcacat 2100 ttgattaatc aacaagttgc tcacgaagtt cttgcgctggaaatcatgat tctgatgctt 2160 gaagaaccaa ctgatgattc agttgaagtc gccattgcgttcctgaaaga gtgtggagca 2220 aagcttctgg agattgctcc agcagctctt aacagtgtctacgaccgtct tcgtgcaatt 2280 ctcatggaaa ctgaaagatc ggaaaatgca ctggatcgacgtattcagta tatgattgag 2340 actgcaatgc agattcgaaa ggacaaattt gcggtaaggtagaatatata aatagtttat 2400 tagaaaaaaa taaattagaa taatttaaat tcctactagccaatcaggcg acctttttgc 2460 gcatagttct attattgaaa aatttggaga atttctcatattctcgctcg gaaatctgga 2520 attcgacgag atcttctggc ttctgtgcag ctgcatcgctttgtgctccc tttctcgctt 2580 gtcttctgtg tacaccaaga accttgttga gttcatcaactgaatctgtg actggcttgt 2640 tgctcactgg atgcactaga cgactgattc tcgagaaatcagattgagtt gcgattaggg 2700 tgacctagaa attgggaata atacgaactt ttgaaaatattcaggaggat taaaaaaatt 2760 attctcgaca atcctacaaa tttacttatt gcaccatgttgctccaacat ttttcattaa 2820 aagttaatga aaaaatgtag aaaatcggaa attggcaattttcagaccat ttttaagcat 2880 tttcaaaaaa aaattgcagc tgaaataaat gtcattttcagataaatcga gcgattttct 2940 gttgtctgac actagttttt agttttaaaa aatgttggaagaacatggtg caataggtaa 3000 tttcatagaa tttccatgtg ttttttttca attaaccaattatccaaatc ttccaaactc 3060 acattttgcg gagctgggct atcaagaatc tgctgcagttttataagacg agcatctctg 3120 atatcactga aaattaattt ttaatcaaaa cttgaatatcaactaaaccc acttattaac 3180 tttctcgatc ttctgtcgtt cggtacgatg acggtgaagaagccaattgt agtagttgat 3240 ttggttcaag tcctttcggt gttgtacgtc agtgtcctgcaatgctattt agttataact 3300 taggcctaag attcaattta atgaagtgat taaatttgttctctgaacct cttaagatga 3360 tcttttggat tagaaacata taagacaggt ttacctatctattaaaaaac agatcaaaat 3420 agatacgacc aaatcggata atccatgcct acctggcatctaggaacgtg ttcttagaag 3480 atttcttacg taatcgtatg aagaaataac aatttgatcgttggccagca aaaatagggt 3540 tttaagtggg atagtgtttt tattagctaa ccggaaaattttatagtttt tttttgcaag 3600 aaaccactga aaacccccta attgtataca ttttttggagcagcttctgg tctttttgag 3660 caataaaatt cgataaaaca gaatttaagt gtaaattgttcacatttagt ttctatttta 3720 tcaaattttg ttgctcaaaa acattcgaag ctgctctaaaaaaatgcatt aaaaaagggg 3780 ttttcagtgg tttttcacat taaaaaagct aattttaactaaaaatccat catatttcca 3840 actttgtcac aacaataaaa tgctggtcaa aatgtgttcgaaaaaatgtt ttttttttta 3900 atttttataa tttaaaaata gttttctttc gctgggacacatacattttt gggcgtaaat 3960 tttcagttca aatttccatt tttacaacca taatcataaagctacgtctg atctctctcg 4020 cacttacctg cgcctgattc gaaagaacaa ccgtagccaaaagaacaaga agaacaagca 4080 cgtagttgtg gtagtggacg ttcatcacgc aatactgaccaatggtcgtg gggtctcact 4140 ttccgtacta ttgagagagg ggagactgaa gatggcaattgaggacagtg tcttcgacgc 4200 acgcatgcat ccataagcat aatccaggag ggatggagagaaaaatcttg tttctaagcc 4260 cctccctttg taatacatac acatatctaa taccgaagaatggctaattg aatggacgtc 4320 agctgttgct gtagttgcca aggcatcatc gatgaaataactgaaagaaa gaattaaata 4380 attattgcag gcgtatccgg cggtcattga agacttggacttgattgagg aggaggatca 4440 gatcatccat acacttaatt tggaggatgc ggttgatccggaaaatgggc ttagtaagtg 4500 actgaccaca cgcggggggc attaatttaa taaattgaattccatttcag atgtgttcaa 4560 actagatcca gaattcgaaa agaacgagga ggtttatgaggagatccgta aggaaatcat 4620 tggaaacgcc gatatttcgg atgaggatgg tggcgacgagttggatgatg aagaagaggg 4680 tagtgatgtg gaagaggctc cgaagaagac tacagagattattgataata ctgatcagaa 4740 ttgactgctt tcagaaggta ttcattttga gttttgggccggcaaatctg taagttgccg 4800 gttgccgaaa atttgctgaa tttgccggaa aaaaaaattccggaatttat ttaaaaactt 4860 tttgtaaaaa ttaaattaaa tttgcaactt ttcagagaagtctacctgac aatgcaatca 4920 tctttggact accaagaagc tgctcacaaa ttgctgaaaatgaagattcc agacagcatg 4980 caggtcagcg atgttgcaaa gaaaaatttt cgaccaaaaaaaccaaccaa tcataaaatt 5040 taaaaaaaaa ctccgttttt ttcttttttt ttatacgagaaaaaccaaaa aaatgtattt 5100 ttgccaaatt ctaaaatact atccccgaaa ttttcaatattttctctttc agaacgaact 5160 ctgcgcgatg cttgtcgatt gttgtgctca acagcgtacctacgagcgat tctacggaat 5220 gctcatcgaa cgtttctgcc gacttcgcct cgaataccagcaatactttg aaaagctctg 5280 ccaggacacg tattccacga ttcaccgaat tgacatcacaaaactgcgga atttggctcg 5340 ccttattgct catttgctct cgacggatgc tattgactggaagattttgg ccgatatgaa 5400 aatgaccgaa gaggacacaa cttcttctgg cagaatctatattaaatata tatttaatga 5460 acttgtggag gcgatgggaa tggttaaact tcattcgagagttactgatc cgtgagtttc 5520 ctagagagag ttgttttcgt attcaatttt ccctattttcagaactttgg ctcattgctt 5580 tgttggatta ttcccacgaa ctaatccgaa cagcgcacgattttcgatca acttcttcac 5640 aatgattgga ttgggtggtt tgacgttgga acttcgtgaatggctggcaa agggtctcaa 5700 gaagaagaag ggaatgctgg atcagttgaa ggccgaatcaagctcagatt catcgtcgtc 5760 ttcggattcg tcagactcgt ctgattcttc ggattctgacgattcatccg actcgtcttc 5820 agattcctca tcttcttcag aatcagagcc agaaccaccgaagaaaaaga agaagaagaa 5880 cagtgaagag agttccaaaa agaaggaaaa agagaatattggtcgacggg atcgtggaga 5940 caagagagct gaacgtcatc gtgatcaaag tgtggagaacaaggacaagg atcgtcgacg 6000 tcgccaggat tctgacgaaa atcgtcggcc agaacgaggagatgaccgca aggatcggag 6060 taaagatcgt cgtcgtcaag actcggatga tgaggatcggaaaggtcgtg aacgtcggga 6120 agattcaggg gaaagacgtc gcggagatcg ggatcgacgtgatcgaaaca aggatcagga 6180 ggatcaccgt gaagatcgcc gtgaccgaag caaggatcgtgaggatcgac gtgatcgccg 6240 tcgtcatgac tctgatgatg atcgtaaaac tcgtcgggatagaagtgaag agcgaggagg 6300 acgtcgtcgt gaagtggaat cggatgatcg acgccgacgtcgttgaattt tcaaatttta 6360 aatactgaat atttgttttt tttcctatta tttatttattctctttgtgt tttttttctt 6420 gctttctaaa aaattaattc aatccaaatc taaacatgagcggttttttt tctctttccg 6480 tctcccaatt cgtattccgc tcctctcatc tgaacacaatgtgcaagttt atttatcttc 6540 tcgctttcat ttcattagga cgtgggggga attggtggaagggggaaaca cacaaaagga 6600 tgatggaaat gaaataagga cacacaatat gcaacaacattcaattcaga aatatggagg 6660 aaggtttaaa agaaaacata aaaatatata gaggaggaaggaaaactagt aaaaaataag 6720 caaagaaatt aggcgaacga tgagaattgt cctcgcttggcaaatgcgaa tccgtatgga 6780 gaggcacgtt tggcgaaggc aaatgttcgg tatggagatctgtaaaaatt tttaagttga 6840 aatttggtgt tgctctttta caaaattttc cgattttcgcttgaaattac ggtgccaggt 6900 ctcgacacgt cttccaattt ttcaaattca aaagagcctttaatgggctg tagttgctaa 6960 tttctcgttt ttgaaaattt ttcttccgtt taatcgaaatttgatgtatt ttatttatga 7020 ttttcaataa atttcaaaga aactggtgaa aactcggaaaattgtgaact acagtaatcc 7080 aatccttaaa ggcgcacacc ttttaaatgt ccgccccaatacgatatttt tttaagattc 7140 gctagagcgg ccgccaccgc ggtggagctc caattcgccctatagtgagt cgtattacaa 7200 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaaccctggcgtta cccaacttaa 7260 tcgccttgca gcacatcccc ccttcgccag ctggcgtaatagcgaagagg cccgcaccga 7320 tcgcccttcc caacagttgc gtagcctgaa tggcgaatgggacgcgccct gtagcggcgc 7380 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgaccgctacacttg ccagcgccct 7440 agcgcccgct cctttcgctt tcttcccttc ctttctcgccacgttcgccg gctttccccg 7500 tcaagctcta aatcgggggc tccctttagg gttccgatttagtgctttac ggcacctcga 7560 ccccaaaaaa cttgattagg gtgatggttc acgtagtgggccatcgccct gatagacggt 7620 ttttcgccct ttgacgttgg agtccacgtt ctttaatagtggactcttgt tccaaactgg 7680 aacaacactc aaccctatct cggtctattc ttttgatttataagggattt tgccgatttc 7740 ggcctattgg ttaaaaaatg agctgattta acaaaaatttaacgcgaatt ttaacaaaat 7800 attaacgttt acaatttcag gtggcacttt tcggggaaatgtgcgcggaa cccctatttg 7860 tttatttttc taaatacatt caaatatgta tccgctcatgagacaataac cctgataaat 7920 gcttcaataa tattgaaaaa ggaagagtat gagtattcaacatttccgtg tcgcccttat 7980 tccctttttt gcggcatttt gccttcctgt ttttgctcacccagaaacgc tggtgaaagt 8040 aaaagatgct gaagatcagt tgggtgcacg agtgggttacatcgaactgg atctcaacag 8100 cggtaagatc cttgagagtt ttcgccccga agaacgttttccaatgatga gcacttttaa 8160 agttctgcta tgtggcgcgg tattatcccg tattgacgccgggcaagagc aactcggtcg 8220 ccgcatacac tattctcaga atgacttggt tgagtactcaccagtcacag aaaagcatct 8280 tacggatggc atgacagtaa gagaattatg cagtgctgccataagcatga gtgataacac 8340 tgcggccaac ttacttctga caacgatcgg aggaccgaaggagctaaccg ctttttttca 8400 caacatgggg gatcatgtaa ctcgccttga tcgttgggaaccggagctga atgaagccat 8460 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatggcaacaacgt tgcgcaaact 8520 attaactggc gaactactta ctctagcttc ccggcaacaattaatagact ggatggaggc 8580 ggataaagtt gcaggaccac ttctgcgctc ggcccttccggctggctggt ttattgctga 8640 taaatctgga gccggtgagc gtgggtctcg cggtatcattgcagcactgg ggccagatgg 8700 taagccctcc cgtatcgtag ttatctacac gacgggcagtcaggcaacta tggatgaacg 8760 aaatagacag atcgctgaga taggtgcctc actgattaagcattggtaac tgtcagacca 8820 agtttactca tatatacttt agattgattt aaaacttcatttttaattta aaaggatcta 8880 ggtgaagatc ctttttgata atctcatgac caaaatcccttaacgtgagt tttcgttcca 8940 ctgagcgtca gaccccgtag aaaagatcaa aggatcttcttgagatcctt tttttctgcg 9000 cgtaatctgc tgcttgcaaa caaaaaaacc accgctaccagcggtggttt gtttgccgga 9060 tcaagagcta ccaactcttt ttccgaaggt aactggcttcagcagagcgc agataccaaa 9120 tactgtcctt ctagtgtagc cgtagttagg ccaccacttcaagaactctg tagcaccgcc 9180 tacatacctc gctctgctaa tcctgttacc agtggctgctgccagtggcg ataagtcgtg 9240 tcttaccggg ttggactcaa gacgatagtt accggataaggcgcagcggt cgggctgaac 9300 ggggggttcg tgcacacagc ccagcttgga gcgaacgacctacaccgaac tgagatacct 9360 acagcgtgag cattgagaaa gcgccacgct tcccgaagggagaaaggcgg acaggtatcc 9420 ggtaagcggc agggtcggaa caggagagcg cacgagggagcttccagggg ggaacgcctg 9480 gtatctttat agtcctgtcg ggtttcgcca cctctgacttgagcgtcgat ttttgtgatg 9540 ctcgtcaggg gggccgagcc tatggaaaaa cgccagcaacgcggcctttt tacggttcct 9600 ggccttttgc tggccttttg ctcacatgtt ctttcctgcgttatcccctg attctgtgga 9660 taaccgtatt accgcctttg agtgagctga taccgctcgccgcagccgaa cgaccgagcg 9720 cagcgagtca gtgagcgagg aagcggaaga gcgcccaatacgcaaaccgc ctctccccgc 9780 gcgttggccg attcattaat gcagctggca cgacaggtttcccgactgga aagcgggcag 9840 tgagcgcaac gcaattaatg tgagttacct cactcattaggcaccccagg ctttacactt 9900 tatgcttccg gctcctatgt tgtgtggaat tgtgagcggataacaatttc acacaggaaa 9960 cagctatgac catgattacg ccaagctcgg aattaaccctcactaaaggg aacaaaagct 10020 gggggggatc ctccaaaatc gtcttccgct ctgaaaaacgaaagtggacc tttgacatcc 10080 gaaaaaatgg gcgaaaaaat gaaattgagc tttttgggtcgaaaaaaatg tttttagaat 10140 gctgagaaca cgttaaacac gaagatcata tttattttgagacccggatg ctctgaaaat 10200 gtctgacata gatttaaaaa agcatatata tatttttcattttcaacgtg aaagttttgt 10260 gcaactttat agaatctcct attggcacat tgttttttatttaactgagg cagtttttga 10320 acaccttttt gaaactttga atctctttga agtatactgtcgaaaagact gacttgagcg 10380 ttcgaaatgc cagaagaaaa ctatatttga atctcgcgctaaattgagaa atgcaaccgc 10440 gctccactgg acaattggaa aaaaaattta ttcggaggcgacaacggtat tttcgaaatt 10500 gattttctgt gtattttctc attttttata aattcttctttgatttatcg ttcgtttgtg 10560 agaaatttaa ttgtattcaa acttttttat agtaagata10599 12 23 DNA Artificial Sequence Description of Artificial SequenceT7 promoter DNA 12 taatacgact cactataggg cga 23 13 35 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide DNA 13agctgtaata cgactcacta tagggcgaga agctt 35 14 35 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide DNA 14 tcgaaagcttctcgcataat agtgagtcgt attac 35 15 29 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide DNA 15 catggcagga tgaacacgattaacatcgc 29 16 32 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide DNA 16 atggccccat ggttacggga acgcgaagtc cg 3217 29 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide DNA 17 atggaattct tacgcgaacg cgaagtccg 29 18 30 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideDNA 18 ctcaccggta atgaacacga ttaacatcgc 30 19 12 PRT Simian virus 40 19Met Thr Ala Pro Lys Lys Lys Arg Lys Val Pro Val 1 5 10 20 37 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideDNA 20 gccaccggtg cgagctcatg aacacgatta acatcgc 37 21 33 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide DNA 21cactagtggg cccttacgcg aacgcgaagt ccg 33 22 33 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide DNA 22 ccggatgactgctccaaaga agaagcgtaa gct 33 23 25 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide DNA 23 cccgggatta atacgactcactata 25 24 33 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide DNA 24 ccggtatagt gagtcgtatt aatcccggga gct 3325 28 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide DNA 25 aattcttaat acgactcact atagggcc 28 26 28 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideDNA 26 catgggccct atagtgagtc gtattaag 28 27 42 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide DNA 27 gatccgtcgacagatctccc tatagtgagt agtattactg ca 42 28 34 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide DNA 28 gtaatacgactcactatagg gagatctgtc gacg 34 29 26 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide DNA 29 tatgccctat agtgagtcgtattaag 26

1. A method for introducing dsRNA into C. elegans, comprising feeding toC. elegans a micro-organism expressing said dsRNA.
 2. A method forintroducing a DNA that encodes and is capable of producing dsRNA into C.elegans, comprising feeding to C. elegans a micro-organism comprisingsaid DNA that encodes and is capable of producing dsRNA.
 3. A methodaccording to claim 2, in which said DNA is in the form of an expressionvector.
 4. A method according to claim 3, in which said expressionvector comprises a promoter or promoters oriented relative to a DNAsequence such that the promoter or promoters initiate transcription ofsaid DNA sequence to double stranded RNA upon binding of a transcriptionfactor to said promoter or promoters.
 5. A method according to claim 4,in which said expression vector comprises two identical promotersflanking said DNA sequence.
 6. A method according to claim 4, in whichsaid expression vector comprises said DNA sequence in a sense and anantisense orientation relative to said promoter or promoters.
 7. Amethod according to claim 4, in which said transcription factor is aphage polymerase.
 8. A method according to claim 7, in which saidpromoter(s) is/are selected from the group consisting of T7, T3 and SP6promoter(s).
 9. A method according to claim 4, in which saidmicro-organism is adapted to express said transcription factor.
 10. Amethod according to claim 9, in which said transcription factor is T7polymerase.
 11. A method according to claim 4, in which said C. elegansis adapted to express said transcription factor.
 12. A method accordingto claim 11, in which said transcription factor is T7 polymerase.
 13. Amethod for down-regulating the expression of a gene of interest in C.elegans, comprising feeding C. elegans with a micro-organism thatexpresses dsRNA corresponding to the gene of interest.
 14. A method fordown-regulating the expression of a gene of interest in C. elegans,comprising feeding C. elegans with a micro-organism that comprises DNAthat encodes and is capable of expressing dsRNA corresponding to thegene of interest in C. elegans.
 15. A method according to claim 14, inwhich said DNA that encodes and is capable of expressing dsRNAcorresponding to the gene of interest is in the form of an expressionvector that comprises a DNA sequence corresponding to the gene ofinterest.
 16. A method according to claim 15, in which said expressionvector comprises a promoter or promoters oriented relative to said DNAsequence such that the promoter or promoters initiate transcription ofsaid DNA sequence to double stranded RNA upon binding of a transcriptionfactor to said promoter or promoters.
 17. A method according to claim16, in which said expression vector comprises two identical promotersflanking said DNA sequence.
 18. A method according to claim 16, in whichsaid expression vector comprises said DNA sequence in a sense and anantisense orientation relative to said promoter or promoters.
 19. Amethod according to claim 16, in which said transcription factor is aphage polymerase.
 20. A method according to claim 19, in which saidpromoter(s) is/are selected from the group consisting of T7, T3 and SP6promoter(s).
 21. A method according to claim 16, in which saidmicro-organism is adapted to express said transcription factor.
 22. Amethod according to claim 21, in which said transcription factor is T7polymerase.
 23. A method according to claim 16, in which said C. elegansis adapted to express said transcription factor.
 24. A method accordingto claim 23, in which said transcription factor is T7 polymerase.
 25. Amethod according to any of claims 1, 2, 13 or 14, in which themicro-organism is a bacterium.
 26. A method according to claim 25, inwhich the bacterium is E. coli.
 27. A method according to claim 26, inwhich the E. coli is a RNAse III negative strain.
 28. A method accordingto any of claims 1, 2, 13 or 14, in which the C. elegans is DNAsedeficient.
 29. A method according to claim 28, in which the DNAsedeficient C. elegans is a nuc-1 mutant.
 30. A micro-organism, comprisingan expression vector that encodes and is capable of producing dsRNA, inwhich said expression vector comprises a promoter or promoters orientedrelative to a DNA sequence such that the promoter or promoters initiatetranscription of said DNA sequence to double stranded RNA upon bindingof a transcription factor to said promoter or promoters.
 31. Amicro-organism according to claim 30, in which said expression vectorcomprises two identical promoters flanking said DNA sequence.
 32. Amicro-organism according to claim 30, in which said expression vectorcomprises said DNA sequence in a sense and an antisense orientationrelative to said promoter or promoters.
 33. A micro-organism accordingto claim 30, in which said transcription factor is a phage polymerase.34. A micro-organism according to claim 33, in which said promoter(s)is/are selected from the group consisting of T7, T3 and SP6 promoter(s).35. A micro-organism according to claim 30, wherein said micro-organismis adapted to express said transcription factor.
 36. A micro-organismaccording to claim 35, wherein said transcription factor is T7polymerase.
 37. A micro-organism according to claim 30, in which saidDNA sequence has been derived from C. elegans.
 38. A micro-organismaccording to claim 37, in which said DNA sequence is a C.elegans-derived cDNA or cDNA fragment.
 39. A micro-organism according toclaim 30, wherein the micro-organism is a bacterium.
 40. Amicro-organism according to claim 39, wherein said bacterium is E. coli.41. A micro-organism according to claim 40, wherein said E.coli_is aRNAse III negative strain.
 42. A library of C. elegans cDNA or C.elegans cDNA fragments, said library having been transformed into amicro-organism by a method comprising: (a) inserting cDNA(s) or cDNAfragment(s) from said C. elegans cDNA library into a cloning site of anexpression vector, in which said expression vector comprises a promoteror promoters oriented relative to said cloning site such that thepromoter or promoters initiate transcription of said cDNA or cDNAfragment inserted in said cloning site to double stranded RNA uponbinding of a transcription factor to said promoter or promoters; (b)transforming the micro-organism with the expression vectors having thecDNA(s) or cDNA fragment(s) inserted therein.
 43. A library according toclaim 42, in which said expression vector comprises two identicalpromoters flanking said cDNA or cDNA fragment.
 44. A library accordingto claim 42, in which said expression vector comprises said cDNA or cDNAfragment in a sense and an antisense orientation relative to saidpromoter or promoters.
 45. A library to according to claim 42, in whichsaid transcription factor is a phage polymerase.
 46. A library accordingto claim 45, in which said promoter(s) is/are selected from the groupconsisting of T7, T3 and SP6 promoter(s).
 47. A library according toclaim 42, in which said micro-organism is adapted to express saidtranscription factor.
 48. A library according to claim 47, in which saidtranscription factor is T7 polymerase.
 49. A library according to claim42, in which the micro-organism is a bacterium.
 50. A library accordingto claim 49, in which the bacterium is E.coli.
 51. A library accordingto claim 50, in which the E. coli is a RNAse III negative strain.
 52. Alibrary according to claim 42, which is organised into hierarchicalpools.
 53. A method for expressing double stranded RNA in C. elegans,comprising feeding C. elegans a library according to any of claims 42,50 and 51.